Methods and compositions for combinatorial barcoding

ABSTRACT

The present disclosure provide compositions, methods and kits for generating a set of combinatorial barcodes, and uses thereof for barcoding samples such as single cells or genomic DNA fragments. Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes. The set of component barcodes can comprise, for example, n×m unique component barcodes, wherein n and m are integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof, wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

The present application is a divisional application of U.S. patent application Ser. No. 15/084,307, filed on Mar. 29, 2016, which claims priority to U.S. Provisional Application No. 62/140,360, filed on Mar. 30, 2015, and U.S. Provisional Application No. 62/152,644, filed on Apr. 24, 2015. The contents of these related applications are hereby expressly incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 68EB-298689-US2, created Nov. 21, 2022, which is 4.0 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

Miniaturization and parallel processing of individual reactions have led to dramatic cost reductions and throughput increases in modern day scientific experiments and measurements. Nucleic acid barcoding can comprise methods wherein the nucleic acids of each sample is distinctively tagged using a sequence string (aka barcode sequence) to add information content to a sequence. Barcoding can comprise separation in chemical (barcode) space, and may or may not depend on physical isolation. DNA barcoding can allow for different sequences to be pooled. DNA barcoding can be difficult to carry out due to the low-throughput of individual tagging with a sample-specific barcode before it can be combined with other barcoded samples. New methods are needed to produce a massive repertoire of barcodes for sample tagging.

SUMMARY

Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes, comprising: n×m unique component barcodes, wherein n and m are positive integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof, wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes. In some embodiments, each one of the set of n unique barcode subunit sequences comprises a molecular label, a cellular label, a dimensional label, a universal label, or any combination thereof. In some embodiments, the total number of different linker sequences is m−1 or m−2. In some embodiments, each of the component barcodes comprises: barcode subunit sequence-linker; complement of linker-barcode subunit sequence-linker; or complement of linker-barcode subunit sequence. In some embodiments, each one of the set of combinatorial barcodes comprises an oligonucleotide comprising the formula: barcode subunit sequences-linkers-barcode subunit sequence_(b)-linker₂- . . . barcode subunit sequence_(c)-linker_(m-1)-barcode subunit sequenced. In some embodiments, the oligonucleotide comprises a target-specific region. In some embodiments, the target-specific region is oligo-dT. In some embodiments, each one of the set of combinatorial barcodes comprises: a first oligonucleotide comprising the formula barcode subunit sequence_(a)-linker₁-barcode subunit sequence_(b)-linker₂- . . . -barcode subunit sequence; and a second oligonucleotide comprising barcode subunit sequence_(d)- . . . -linker₃-barcode subunit sequence_(e)-linker_(m-2)-barcode subunit sequence_(f). In some embodiments, each of the first oligonucleotide and the second oligonucleotide comprises a target-specific region. In some embodiments, one of the target-specific regions is oligo-dT. In some embodiments, each one of the one or two linker sequences is different. In some embodiments, m is an integer from 2 to 10. In some embodiments, m is an integer from 2 to 4. In some embodiments, n is an integer from 4-100. In some embodiments, n=24. In some embodiments, the n unique barcode subunit sequences are the same length. In some embodiments, at least two of the n unique barcode subunit sequences have different lengths. In some embodiments, the linker sequences are the same length. In some embodiments, the linker sequences have different lengths. In some embodiments, the set of combinatorial barcodes has equal to or less than n^(m) unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 100,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 200,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 300,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 400,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 1,000,000 unique combinatorial barcodes.

Some embodiments disclosed herein provide methods of barcoding a plurality of partitions comprising: introducing m component barcodes in each of the plurality of partitions, wherein the m component barcodes are selected from a set of n×m different component barcodes, wherein n and m are positive integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof; and connecting the m component barcodes to generate a combinatorial barcode, whereby each of the plurality of partitions is associated with a unique combinatorial barcode comprising the m component barcodes. In some embodiments, each of the plurality of the partitions comprises a sample. In some embodiments, the sample is a DNA sample. In some embodiments, the sample is an RNA sample. In some embodiments, the sample is a single cell. In some embodiments, the total number of different linker sequences is m−1 or m−2. In some embodiments, the methods comprise hybridizing one or two of the m component barcodes to a target of the sample. In some embodiments, the methods comprise extending the one or two of the m component barcodes hybridized to the target, whereby the target is labeled with the combinatorial barcode. In some embodiments, the component barcodes are introduced in each of the plurality of partitions using an inkjet printer.

Some embodiments disclosed herein provide methods of barcoding a plurality of DNA targets in a plurality of partitions comprising: depositing m component barcodes in each of the plurality of partitions comprising a DNA target, wherein the m component barcodes are selected from a set of n×m different component barcodes, wherein n and m are positive integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof; and connecting the m component barcodes to generate a combinatorial barcode, whereby the DNA target in each of the plurality of partitions is associated with a unique combinatorial barcode comprising the m component barcodes. In some embodiments, the total number of different linker sequences is m−1 or m−2. In some embodiments, the methods comprise hybridizing one or two of the m component barcodes to the DNA target. In some embodiments, the methods comprise extending the one or two of the m component barcodes hybridized to the DNA target to produce an extension product comprising the combinatorial barcode. In some embodiments, the methods comprise amplifying the extension product. In some embodiments, the amplifying the extension product comprises isothermal multiple strand displacement amplification. In some embodiments, the methods comprise pooling the amplification products from the plurality of partitions. In some embodiments, the methods comprise sequencing the pooled amplification products. In some embodiments, the methods comprise assembling the sequences using the combinatorial barcodes. In some embodiments, the methods comprise generating haplotypes using the assembled sequences. In some embodiments, the haplotypes are generated using overlapping sequencing reads that cover at least 100 kb.

Some embodiments disclosed herein provide methods of single cell sequencing comprising: depositing m component barcodes in each of a plurality of partitions comprising a single cell, wherein the m component barcodes are selected from a set of n×m different component barcodes, wherein n and m are positive integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof; and connecting the m component barcodes to generate a combinatorial barcode, whereby a target of the single cell in each of the plurality of partitions is associated with a unique combinatorial barcode comprising the m component barcodes. In some embodiments, the total number of different linker sequences is m−1 or m−2. In some embodiments, the target is a DNA. In some embodiments, the target is an RNA. In some embodiments, the methods comprise hybridizing one or two of the m component barcodes to the target. In some embodiments, the methods comprise extending the one or two of the m component barcodes hybridized to the target to produce an extension product comprising the combinatorial barcode. In some embodiments, the methods comprise amplification of the extension product. In some embodiments, the methods comprise pooling the amplification products from the plurality of partitions. In some embodiments, the methods comprise sequencing the pooled amplification products. In some embodiments, the methods comprise assembling the sequences from a single cell using the combinatorial barcodes. In some embodiments, the methods comprise massively parallel characterization of single cells.

Some embodiments disclosed herein provide methods of spatially barcoding a target in a sample, comprising: providing a plurality of oligonucleotides immobilized on a substrate; contacting a sample with the plurality of oligonucleotides immobilized on the substrate; hybridizing the target with a probe that specifically binds to the target; capturing an image showing the locations of the target; capturing an image of the sample; and correlating the image showing the locations of the target and the image of the sample to spatially barcode the target in the sample. In some embodiments, the oligonucleotides immobilized on the substrate are oligo-dT primers. In some embodiments, the oligonucleotides immobilized on the substrate are random primers. In some embodiments, the oligonucleotides immobilized on the substrate are target-specific primers. In some embodiments, the methods comprise reverse transcribing the target to generate a cDNA. In some embodiments, the methods comprise adding dATP homopolymers to the cDNA. In some embodiments, the methods comprise amplifying the cDNA using bridge amplification. In some embodiments, the substrate is part of a flow cell. In some embodiments, the methods comprise lysing the sample before or after contacting the sample with the plurality of oligonucleotides immobilized on the substrate. In some embodiments, lysing the sample comprises heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof. In some embodiments, the sample comprises a tissue slice, a cell monolayer, fixed cells, a tissue section, or any combination thereof. In some embodiments, the sample comprises a plurality of cells. In some embodiments, the plurality of cells comprises one or more cell types. In some embodiments, at least one of the one or more cell types is brain cell, heart cell, cancer cell, circulating tumor cell, organ cell, epithelial cell, metastatic cell, benign cell, primary cell, circulatory cell, or any combination thereof. In some embodiments, the methods comprise amplifying the target to generate a plurality of amplicons immobilized on the substrate. In some embodiments, the target comprises ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs, small interfering RNAs (siRNAs), RNA degradation products, RNAs each comprising a poly(A) tail, or any combination thereof. In some embodiments, the methods comprise spatially barcoding a plurality of targets. In some embodiments, the probe is 18-100 nt long. In some embodiments, the probe comprises a fluorescent label. In some embodiments, the image showing the locations of the target is a fluorescent image. In some embodiments, the sample comprises an immunohistochemical staining, a progressive staining, a hematoxylin-eosin staining, or a combination thereof. In some embodiments, the solid support comprises a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof.

Some embodiments disclosed herein provide methods of single cell sequencing comprising: depositing a first oligonucleotide comprising a first barcode selected from a first set of 40 unique barcodes and a first primer into a plurality of partitions comprising a single cell; depositing a second oligonucleotide comprising a second barcode selected from a second set of 40 unique barcodes and a second primer into the plurality of partitions comprising the single cell; contacting the first oligonucleotide with a target from the single cell; extending the first oligonucleotide to generate a first strand comprising the first barcode; contacting the second oligonucleotide with the first strand; and extending the second oligonucleotide to generate a second strand comprising the first barcode and the second barcode, whereby the target from the single cell is labeled with the first barcode and the second barcode. In some embodiments, the methods comprise amplifying the second strand to produce amplification products. In some embodiments, the methods comprise pooling the amplification products from the plurality of partitions. In some embodiments, the methods comprise sequencing the pooled amplification products. In some embodiments, the methods comprise assembling the sequences from a single cell using the first barcode and the second barcode. In some embodiments, the methods comprise massively parallel characterization of single cells. In some embodiments, the plurality of partitions is comprised of a 1536 microwell plate. In some embodiments, the target is an mRNA. In some embodiments, the first primer is an oligo-dT primer. In some embodiments, the target is a DNA.

Some embodiments disclosed herein provide compositions comprising a set of combinatorial barcodes, wherein each combinatorial barcode of the set of combinatorial barcodes comprises: m barcode subunit sequences, each selected from a set of n unique barcode subunit sequences, wherein n and m are positive integers; and m−1 or m−2 linker sequences, wherein the m barcode subunit sequences are connected to each other through the m−1 or m−2 linker sequences, and the total number of unique combinatorial barcodes in the set of combinatorial barcodes is more than 384. In some embodiments, each one of the set of n unique barcode subunit sequences comprises a molecular label, a cellular label, a dimensional label, a universal label, or any combination thereof. In some embodiments, the total number of unique combinatorial barcodes in the set of combinatorial barcodes is equal to or less than n^(m). In some embodiments, each one of the set of combinatorial barcodes comprises an oligonucleotide comprising the formula: barcode subunit sequences-linkers-barcode subunit sequence_(b)-linker₂- . . . barcode subunit sequence_(c)-linker_(m-1)-barcode subunit sequence_(d). In some embodiments, each one of the set of combinatorial barcodes comprises: a first oligonucleotide comprising the formula barcode subunit sequence_(a)-linker₁-barcode subunit sequence_(b)-linker₂- . . . -barcode subunit sequence; and a second oligonucleotide comprising barcode subunit sequence_(d)- . . . -linker₃-barcode subunit sequence_(e)-linker_(m-2)-barcode subunit sequence_(f). In some embodiments, each one of the set of combinatorial barcodes comprises one or two target-specific regions. In some embodiments, one of the one or two target-specific regions is oligo-dT. In some embodiments, each one of the m−1 or m−2 linker sequences is different. In some embodiments, m is an integer from 2 to 10. In some embodiments, m is an integer from 2 to 4. In some embodiments, n is an integer from 4-100. In some embodiments, n=24. In some embodiments, the n unique barcode subunit sequences are the same length. In some embodiments, the n unique barcode subunit sequences have different lengths. In some embodiments, the m−1 or m−2 linker sequences are the same length. In some embodiments, the m−1 or m−2 linker sequences have different lengths. In some embodiments, the set of combinatorial barcodes has at least 100,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 200,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 300,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 400,000 unique combinatorial barcodes. In some embodiments, the set of combinatorial barcodes has at least 1,000,000 unique combinatorial barcodes. In some embodiments, each one of the set of combinatorial barcodes is attached to a solid support. In some embodiments, each one of the set of combinatorial barcodes is used to label a partition. In some embodiments, the partition is part of a microwell array having more than 10,000 microwells. In some embodiments, each microwell of the microwell array comprises a different combinatorial barcode from the set of combinatorial barcodes.

In one aspect the disclosure provides for a composition comprising: a set of reagent barcodes, wherein each reagent barcode m has a length of n, wherein the number of different reagent barcodes is n×m, and wherein the possible number of different combinatorial barcodes is n^(m). In some embodiments, the different combinatorial barcodes are combinations of the reagent barcodes. In some embodiments, the different combinatorial barcodes differ by at least 1 nucleotide. In some embodiments, the different combinatorial barcodes differ by at least 2 nucleotides. In some embodiments, n^(m)/(n×m) is at least 1:2000. In some embodiments, n^(m)/(n×m) is at least 1:3000. In some embodiments, n^(m)/(n×m) is at least 1:4000. In some embodiments, wherein the number of different combinatorial barcodes is at least 100,000. In some embodiments, the number of the different combinatorial barcodes is at least 200,000. In some embodiments, the number of the different combinatorial barcodes is at least 300,000. In some embodiments, the number of the different combinatorial barcodes is at least 400,000. In some embodiments, the number of the different combinatorial barcodes is 331,776. In some embodiments, m is at least 2. In some embodiments, m is at least 3. In some embodiments, m is at least 4. In some embodiments, m is 4. In some embodiments, n is from 5-50. In some embodiments, n is 24. In some embodiments, reagent barcodes of the set of reagent barcodes comprise different lengths. In some embodiments, a first barcode is linked to a second barcode of the set of barcodes through a linker. In some embodiments, the first barcode comprises a target-specific region. In some embodiments, the target-specific region hybridizes to a sense strand of a target polynucleotide. In some embodiments, the combinatorial barcode is bipartite. In some embodiments, the combinatorial barcode is tripartite. In some embodiments, the combinatorial code is partially on the 3′ end of a target polynucleotide and partially on the 5′ end of the target polynucleotide. In some embodiments, the target-specific region hybridizes to an anti-sense strand of a target polynucleotide. In some embodiments, the composition further comprises a target polynucleotide.

In one aspect the disclosure provides for: a method for barcoding samples comprising: contacting a set of reagent barcodes, wherein each reagent barcode m has a length of n, wherein the number of different reagent barcodes is n×m, and wherein the possible number of different combinatorial barcodes is e to a plurality of partitions; associating the barcodes reagents with target polynucleotides; labelling the target polynucleotides with the barcode reagents, wherein each of the target polynucleotides comprises a different combinatorial barcode. In some embodiments, the target polynucleotides are DNA. In some embodiments, the target polynucleotides are genomic DNA. In some embodiments, the target polynucleotides are RNA. In some embodiments, the target polynucleotides are fragments of chromosomes. In some embodiments, the target polynucleotides are at least 100 kilobases in length. In some embodiments, the target polynucleotides are from a single cell.

In one aspect, the disclosure provides for a method for isolating a plurality of cells comprising: isolating a single cell of the plurality of cells into a single well a substrate in a non-Poisson manner, wherein the well comprises two or more combinatorial barcode reagents; and labeling a nucleic acid from the cell with the combinatorial barcode reagents, thereby generating a combinatorially barcoded nucleic acid. In some embodiments, the isolating comprises distributing the plurality of cells such that at least 20% of wells of the substrate have a single cell. In some embodiments, the isolating comprises distributing the plurality of cells such that at least 40% of wells of the substrate have a single cell. In some embodiments, the isolating comprises distributing the plurality of cells such that at least 60% of wells of the substrate have a single cell. In some embodiments, the isolating comprises distributing the plurality of cells such that at least 80% of wells of the substrate have a single cell. In some embodiments, the isolating comprises distributing the plurality of cells such that 100% of wells of the substrate have a single cell. In some embodiments, the isolating is non-random. In some embodiments, the isolating is performed by an isolating device. In some embodiments, the isolating device comprises a device selected from the group consisting of: a flow cytometer, a needle array, and a microinjector, or any combination thereof. In some embodiments, the substrate comprises at least 500 wells. In some embodiments, the substrate comprises at least 1,000 wells. In some embodiments, the substrate comprises at least 10,000 wells. In some embodiments, the substrate comprises 96, 384, or 1536 wells. In some embodiments, a combinatorial barcode reagent of the combinatorial barcode reagents comprises a subunit code section. In some embodiments, the combinatorial barcode reagent further comprises a linker adjacent to the subunit code section. In some embodiments, the linker is located 5′, 3′ or both 5 and 3′ to the subunit code section. In some embodiments, linkers from different combinatorial barcode reagents are configured to hybridize together, thereby generating concatenated combinatorial barcode reagents. In some embodiments, the concatenated barcode reagents correspond to a combinatorial barcode. In some embodiments, the combinatorial barcode is bipartite. In some embodiments, the combinatorial barcode is partially on the 3′ end of the combinatorially barcoded nucleic acid and partially on the 5′ end of the combinatorially barcoded nucleic acid. In some embodiments, the subunit code section is from 4-30 nucleotides in length. In some embodiments, the subunit code section is 6 nucleotides in length. In some embodiments, each subunit code section of a combinatorial barcode reagent comprises a unique subunit code sequence. In some embodiments, a combinatorial barcode reagent of the combinatorial barcode reagents comprises a target-specific region. In some embodiments, only one of the combinatorial barcode reagents comprises a target-specific region. In some embodiments, only two of the combinatorial barcode reagents comprises a target-specific region. In some embodiments, the target-specific region is selected from the group consisting of: a random multimer, oligo dT, or a gene-specific sequence. In some embodiments, the target-specific region is adapted to hybridize to the sense strand of a target nucleic acid. In some embodiments, target-specific region is adapted to hybridize to the anti-sense strand of a target nucleic acid. In some embodiments, the labelling comprises hybridizing the combinatorial barcode reagents to the nucleic acids. In some embodiments, the hybridizing further comprises hybridizing combinatorial barcodes reagents to each other through their cognate linkers. In some embodiments, the method further comprises extending the combinatorial barcode reagents. In some embodiments, the extending comprises primer extension of the combinatorial barcode reagents. In some embodiments, the extending comprises generating a transcript comprising the sequence of the combinatorial barcode reagents and the nucleic acid. In some embodiments, wells of the substrate comprise different combinations of the combinatorial barcode reagents. In some embodiments, the different combinations of the combinatorial barcode reagents correspond to different combinatorial barcodes. In some embodiments, the method further comprises pooling the combinatorially barcoded nucleic acids after the labelling. In some embodiments, the method further comprises amplifying the combinatorially barcoded nucleic acids, thereby generating a combinatorially barcoded amplicon. In some embodiments, the amplifying comprises multiple strand displacement. In some embodiments, the amplifying comprises PCR. In some embodiments, the amplifying is performed with primers that hybridize to sequences the combinatorial barcode reagents. In some embodiments, the amplifying is performed with a gene-specific primer and a primer that hybridizes to a sequence of a combinatorial barcode reagent. In some embodiments, the method further comprises determining the sequence of the combinatorially barcoded amplicon. In some embodiments, the determining comprises determining a portion of the sequence of the combinatorial barcode of the combinatorially barcoded amplicon and a portion of the sequence of the nucleic acid of the combinatorially barcoded amplicon. In some embodiments, the method further comprises lysing the cells prior to the labeling. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is genomic DNA. In some embodiments, the cells comprise a cell selected from the group consisting of: a human cell, a mammalian cell, a rat cell, a pig cell, a mouse cell, a fly cell, a worm cell, an invetebrate cell, a vertebrate cell, a fungi cell, a bacterial cell, and a plant cell, or any combination thereof. In some embodiments, the cells comprise a tumor cell. In some embodiments, the cells comprise a diseased cell. In some embodiments, the number of the combinatorial barcode reagents in the well is at least 2. In some embodiments, the number of the combinatorial barcode reagents in the well is at least 3. In some embodiments, the number of the combinatorial barcode reagents in the well is at least 4. In some embodiments, the number of the combinatorial barcode reagents in the well is 4.

In one aspect the disclosure provides for a kit comprising: a set of combinatorial barcode reagents, wherein only one of the combinatorial barcode reagents comprises a target-specific sequence, wherein barcodes of the set of combinatorial barcode reagents comprise linkers such that the combinatorial barcode reagents overlap through the linkers, thereby generating combinatorial barcodes. In some embodiments, the combinatorial barcode comprises a combination of the combinatorial barcode reagents. In some embodiments, the sequences of the combinatorial barcodes differ by at least 1 nucleotide. In some embodiments, the sequences of the combinatorial barcodes differ by at least 2 nucleotides. In some embodiments, the number of the combinatorial barcodes generated from the set of combinatorial barcode reagents is at least 100,000. In some embodiments, the number of the combinatorial barcodes generated from the set of combinatorial barcode reagents is at least 200,000. In some embodiments, the number of the combinatorial barcodes generated from the set of combinatorial barcode reagents is at least 300,000. In some embodiments, the number of the combinatorial barcodes generated from the set of combinatorial barcode reagents is at least 400,000. In some embodiments, the number of the combinatorial barcodes generated from the set of combinatorial barcode reagents is 331,776. In some embodiments, a combinatorial barcode reagent of the combinatorial barcode reagents comprises a subunit code section. In some embodiments, the combinatorial barcode reagent further comprises a linker adjacent to the subunit code section. In some embodiments, the linker is located 5′, 3′ or both 5 and 3′ to the subunit code section. In some embodiments, linkers from different combinatorial barcode reagents are configured to hybridize together, thereby generating concatenated combinatorial barcode reagents. In some embodiments, the concatenated barcode reagents comprise a combinatorial barcode. In some embodiments, the subunit code section is from 5-35 nucleotides in length. In some embodiments, the subunit code section is 6 nucleotides in length. In some embodiments, each subunit code section of a combinatorial barcode comprises a unique subunit code sequence. In some embodiments, each reagent barcode m has a subunit code length of n, and wherein n^(m)/(n×m) is at least 2000. In some embodiments, n^(m)/(n×m) is at least 3000. In some embodiments, n^(m)/(n×m) is at least 4000. In some embodiments, n×m is the number of different reagent barcodes. In some embodiments, n^(m) is the possible number of different combinatorial barcodes. In some embodiments, a combinatorial barcode reagent of the combinatorial barcode reagents comprises a target-specific region. In some embodiments, the target-specific region is selected from the group consisting of: a random multimer, oligo dT, or a gene-specific sequence. In some embodiments, the target-specific region is adapted to hybridize to the sense strand of a target nucleic acid. In some embodiments, target-specific region is adapted to hybridize to the anti-sense strand of a target nucleic acid. In some embodiments, the combinatorial barcode is bipartite. In some embodiments, a portion of the combinatorial barcode is partially on the 3′ end of a target polynucleotide and partially on the 5′ end of the target polynucleotide. In some embodiments, wherein the kit further comprises a substrate. In some embodiments, the substrate comprises at least 1,000 microwells. In some embodiments, the microwells comprise at least two reagent barcodes of the set of reagent barcodes. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit further comprises a buffer. In some embodiments, the kit further comprises a gene-specific primer. In some embodiments, the kit further comprises amplification primers.

In one aspect, the disclosure provides for a method for whole genome sequencing comprising: fragmenting a nucleic acid chromosome into one or more chromosomal fragments; isolating the one or more chromosomal fragments into a well of a substrate comprising two or more reagent barcodes; amplifying the chromosomal fragments with the reagent barcodes, thereby generating combinatorially barcoded fragments; and determining the sequence of the combinatorially barcoded fragments, thereby performing whole genome sequencing. In some embodiments, the chromosomal fragments are at least 100 kilobases in length. In some embodiments, the chromosomal fragments are from 50 to 300 kilobases in length. In some embodiments, the fragmenting produces from 1×10⁶ to 1×10⁷ fragments. In some embodiments, the isolating comprises isolating from 1-5 fragments in the well. In some embodiments, the 1-5 fragments are non-overlapping. In some embodiments, the amplifying comprises multiple strand displacement. In some embodiments, the amplifying is isothermal. In some embodiments, the method further comprises pooling the combinatorially barcoded fragments prior to the determining. In some embodiments, the determining comprises sequencing at least a portion of the combinatorial barcode and at least a portion of the chromosomal fragment. In some embodiments, the determining comprises grouping sequence reads into groups based on their combinatorial barcode sequence. In some embodiments, the groups correspond to wells of the substrate. In some embodiments, the method further comprises assembling contigs from the chromosomal fragments. In some embodiments, the method further comprises mapping the contigs to a maternal or paternal chromosome. In some embodiments, the mapping determines haplotype phasing of the chromosomal fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 illustrates an exemplary embodiment of the combinatorial barcode reagents of the disclosure.

FIG. 2 illustrates exemplary embodiments of concatenation of combinatorial barcode reagents of the disclosure

FIG. 3 illustrates an exemplary embodiment of the methods of the disclosure for combinatorial barcoding of nucleic acids.

FIG. 4 depicts an exemplary embodiment of the method of generating contigs using combinatorial barcode reagents of the disclosure.

FIG. 5 depicts an exemplary embodiment of the method of genotyping single cells using combinatorial barcode reagents of the disclosure.

FIG. 6 illustrates an exemplary embodiment of method of combining combinatorial barcoding and stochastic barcoding methods of the disclosure.

FIG. 7 illustrates an exemplary embodiment of a bipartite combinatorial barcode method of the disclosure.

FIG. 8 illustrates an exemplary embodiment of a combinatorial barcode immobilized on a solid support.

FIG. 9 illustrates an exemplary embodiment of the methods of the disclosure for spatially barcoding s target in a sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The disclosure provides compositions and methods for production of a repertoire of diverse barcoding regents using only a small set of component barcode reagents. The method of the disclosure provides for simple molecular biology steps to carry out the combinatorial pairing of the component barcoding reagents. In some instances, hundreds of thousands to over millions of samples can be processed in parallel. For example, the number of samples that can be processed in parallel can be at least 100, 1,000, 5,000, 10,000, 50,000, 100,000, 200,000, 300,000; 400,000, 500,000, 600,000, 700,000, 800,000, or 900,000. In some embodiments, the number of samples that can be processed in parallel can be at most 100, 1,000, 5,000, 10,000, 50,000, 100,000, 200,000, 300,000; 400,000, 500,000, 600,000, 700,000, 800,000, or 900,000. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more millions of samples can be processed in parallel. In some embodiments, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more millions of samples can be processed in parallel.

The disclosure provides for methods, compositions, kits, and systems for combinatorial barcoding of nucleic acids using combinatorial barcode reagents. As shown in FIG. 1 , a combinatorial barcode reagent of the disclosure 105/110/115/120 can comprise a target-specific region, a subunit code section, and a linker region, or any combination thereof. The subunit code section XXXXXX can have a subunit code sequence. Different subunit code sections can have different subunit code sequences. As shown in FIG. 1 , combinatorial barcode reagents 105, 110, 115, and 120 can be concatenated together through linker regions. Concatenated combinatorial barcode reagents can be extended (e.g., with primer extension) to generate a transcript 125 comprising the sequences of the combinatorial barcode reagents and the target polynucleotide. Combinatorial barcode reagents can reduce the number reagents necessary for generating a massive repertoire of barcodes.

Definitions

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

As used herein the term “associated” or “associated with” can mean that two or more species are identifiable as being co-located at a point in time. An association can mean that two or more species are or were within a similar container. An association can be an informatics association, where for example digital information regarding two or more species is stored and can be used to determine that one or more of the species were co-located at a point in time. An association can also be a physical association. In some instances two or more associated species are “tethered”, “attached”, or “immobilized” to one another or to a common solid or semisolid surface. An association may refer to covalent or non-covalent means for attaching labels to solid or semi-solid supports such as beads. An association may comprise hybridization between a target and a label.

The terms “component barcode,” “reagent barcode” and “combinatorial barcode reagent” are used interchangeably to refer to a polynucleotide sequence that comprises a subunit code sequence, and can be used with one or more other combinatorial barcode reagents to generate combinatorial barcodes. For example, combinatorial barcode reagents can be concatenated through linkers, thereby generating a combinatorial barcode.

As used herein, the term “combinatorial barcode” refers to a polynucleotide sequence that comprises the sequence of one or more combinatorial barcode reagents.

As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be the “complement” of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be said to be the “reverse complement” of a second sequence, if the first nucleotide sequence is complementary to a sequence that is the reverse (i.e., the order of the nucleotides is reversed) of the second sequence. As used herein, the terms “complement”, “complementary”, and “reverse complement” can be used interchangeably. It is understood from the disclosure that if a molecule can hybridize to another molecule it may be the complement of the molecule that is hybridizing.

As used herein, the term “digital counting” refers to a method for estimating a number of target molecules in a sample. Digital counting can include the step of determining a number of unique labels that have been associated with targets in a sample. This stochastic methodology transforms the problem of counting molecules from one of locating and identifying identical molecules to a series of yes/no digital questions regarding detection of a set of predefined labels.

As used herein, the term “first universal label” refers to a label that is universal for barcodes of the disclosure. A first universal label can be a sequencing primer binding site (e.g., a read primer binding site, i.e., for an Illumina sequencer).

As used herein, the term “label” or “labels” refers to nucleic acid codes associated with a target within a sample. A label can be, for example, a nucleic acid label. A label can be an entirely or partially amplifiable label. A label can be entirely or partially sequencable label. A label can be a portion of a native nucleic acid that is identifiable as distinct. A label can be a known sequence. A label can comprise a junction of nucleic acid sequences, for example a junction of a native and non-native sequence. As used herein, the term “label” can be used interchangeably with the terms, “index”, “tag,” or “label-tag.” Labels can convey information. For example, in various embodiments, labels can be used to determine an identity of a sample, a source of a sample, an identity of a cell, and/or a target.

As used herein, the term “non-depleting reservoirs” refers to a pool of stochastic barcodes made up of many different labels. A non-depleting reservoir can comprise large numbers of different stochastic barcodes such that when the non-depleting reservoir is associated with a pool of targets each target is likely to be associated with a unique stochastic barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random choice, and depends on the number of copies of identical target molecules in the collection compared to the diversity of labels. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and analysis of the number of stochastic barcodes detected then allows calculation of the number of target molecules present in the original collection or sample. When the ratio of the number of copies of a target molecule present to the number of unique stochastic barcodes is low, the labeled target molecules are highly unique (i.e. there is a very low probability that more than one target molecule will have been labeled with a given label).

As used herein, a “nucleic acid” refers to a polynucleotide sequence, or fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or fragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one or more analogs (e.g. altered backgone, sugar, or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or flurescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. “Nucleic acid”, “polynucleotide, “target polynucleotide”, and “target nucleic acid” can be used interchangeably.

A nucleic acid can comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). A nucleic acid can comprise a nucleic acid affinity tag. A nucleoside can be a base-sugar combination. The base portion of the nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within nucleic acids, the phosphate groups can commonly be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone of the nucleic acid can be a 3′ to 5′ phosphodiester linkage.

A nucleic acid can comprise a modified backbone and/or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, a 5′ to 5′ or a 2′ to 2′ linkage.

A nucleic acid can comprise polynucleotide backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These can include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

A nucleic acid can comprise a nucleic acid mimetic. The term “mimetic” include, for example, polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. The backbone in PNA compounds can comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

A nucleic acid can comprise a morpholino backbone structure. For example, a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace a phosphodiester linkage.

A nucleic acid can comprise linked morpholino units (i.e. morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomeric units in a morpholino nucleic acid. Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. A variety of compounds within the morpholino class can be joined using different linking groups. A further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a nucleic acid molecule can be replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. The incorporation of CeNA monomers into a nucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes. A further modification can include Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (—CH2-), group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs can display very high duplex thermal stabilities with complementary nucleic acid (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation and good solubility properties.

A nucleic acid can also, in some embodiments, include nucleobase (often referred to simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases can include the purine bases, (e.g. adenine (A) and guanine (G)), and the pyrimidine bases, (e.g. thymine (T), cytosine (C) and uracil (U)). Modified nucleobases can include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (Hpyrido(3′,′:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

As used herein, the term “sample” refers to a composition comprising targets. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, single cells, tissues, organs, or organisms.

As used herein, the term “sampling device” or “device” refers to a device which may take a section of a sample and/or place the section on a substrate. A sample device can refer to, for example, a fluorescence activated cell sorting (FACS) machine, a cell sorter machine, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and/or a microtome.

As used herein, the term “solid support” refers to discrete solid or semi-solid surfaces to which a plurality of stochastic barcodes may be attached. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently). A solid support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. A plurality of solid supports spaced in an array may not comprise a substrate. A solid support may be used interchangeably with the term “bead.”

A solid support can refer to a “substrate.” A substrate can be a type of solid support. A substrate can refer to a continuous solid or semi-solid surface on which the methods of the disclosure may be performed. A substrate can refer to an array, a cartridge, a chip, a device, and a slide, for example. As used herein, “solid support” and “substrate” can be used interchangeably.

As used herein, the term “stochastic barcode” refers to a polynucleotide sequence comprising labels of the present disclosure. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding. Stochastic barcodes can be used to quantify targets within a sample. Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target. For example, a stochastic barcode can be used to assess amplification or sequencing errors. A stochastic barcode associated with a target can be called a stochastic barcode-target or stochastic barcode-tag-target.

As used herein, the term “stochastic barcoding” refers to the random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a recursive Poisson strategy to associate and quantify labels associated with targets. As used herein, the term “stochastic barcoding” can be used interchangeably with “stochastic labeling.”

As used here, the term “target” refers to a composition which can be associated with a stochastic barcode. Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. Targets can be single or double stranded. In some embodiments targets can be proteins. In some embodiments targets are lipids.

The term “reverse transcriptases” refers to a group of enzymes having reverse transcriptase activity (i.e., that catalyze synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptase, and mutants, variants or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transciptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include the Lactococcus lactis L1.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases can include many classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity-generating retroelements among others).

The term “template switching” refers to the ability of a reverse transcriptase to switch from an initial nucleic acid sequence template to the 3′ end of a new nucleic acid sequence template having little or no complementarity to the 3′ end of the nucleic acid synthesized from the initial template. Nucleic acid copies of a target polynucleotide can be made using template switching. Template switching allows, e.g., a DNA copy to be prepared using a reverse transcriptase that switches from an initial nucleic acid sequence template to the 3′ end of a new nucleic acid sequence template having little or no complementarity to the 3′ end of the DNA synthesized from the initial template, thereby allowing the synthesis of a continuous product DNA that directly links an adaptor sequence to a target oligonucleotide sequence without ligation. Template switching can comprise ligation of adaptor, homopolymer tailing (e.g., polyadenylation), random primer, or an oligonucleotide that the polymerase can associate with.

Stochastic Barcodes

As disclosed herein, a stochastic barcode can be a polynucleotide sequence that may be used to stochastically label (e.g., barcode, tag) a target. A stochastic barcode can comprise one or more labels. Exemplary labels include, but are not limited to, universal labels, cellular labels, molecular labels, sample labels, plate labels, spatial labels, pre-spatial labels, and any combination thereof. A stochastic barcode can comprise a 5′amine that may link the stochastic barcode to a solid support. The stochastic barcode can comprise one or more of a universal label, a dimension label, a spatial label, a cellular label, and a molecular label. The universal label may be 5′-most label. The molecular label may be the 3′-most label. The spatial label, dimension label, and the cellular label may be in any order. In some instances, the universal label, the spatial label, the dimension label, the cellular label, and the molecular label are in any order. The stochastic barcode can comprise a target-binding region. The target-binding region can interact with a target (e.g., target nucleic acid, RNA, mRNA, DNA) in a sample. For example, a target-binding region can comprise an oligo dT sequence which can interact with poly-A tails of mRNAs. In some instances, the labels of the stochastic barcode (e.g., universal label, dimension label, spatial label, cellular label, and molecular label) may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides.

A stochastic barcode can comprise one or more universal labels. The one or more universal labels can be the same for all stochastic barcodes in the set of stochastic barcodes (e.g., attached to a given solid support). In some embodiments, the one or more universal labels can be the same for all stochastic barcodes attached to a plurality of beads. In some embodiments, a universal label comprises a nucleic acid sequence that is capable of hybridizing to a sequencing primer. Sequencing primers can be used for sequencing stochastic barcodes comprising a universal label. Sequencing primers (e.g., universal sequencing primers) can comprise sequencing primers associated with high-throughput sequencing platforms. In some embodiments, a universal label may comprise a nucleic acid sequence that is capable of hybridizing to a PCR primer. In some embodiments, the universal label comprises a nucleic acid sequence that is capable of hybridizing to a sequencing primer and a PCR primer. The nucleic acid sequence of the universal label that is capable of hybridizing to a sequencing or PCR primer may be referred to as a primer binding site. A universal label can comprise a sequence that may be used to initiate transcription of the stochastic barcode. A universal label can comprise a sequence that may be used for extension of the stochastic barcode or a region within the stochastic barcode. A universal label can be, or be at least about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. A universal label can comprise at least about 10 nucleotides. A universal label can be at most about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, a cleavable linker or modified nucleotide is part of the universal label sequence to enable the stochastic barcode to be cleaved off from the support. As used herein, a universal label can be used interchangeably with “universal PCR primer.”

A stochastic barcode can comprise a dimension label. A dimension label can comprise a nucleic acid sequence that provides information about a dimension in which the stochastic labeling occurred. For example, a dimension label can provide information about the time at which a target was stochastically barcoded. A dimension label can be associated with a time of stochastic barcoding in a sample. A dimension label can activated at the time of stochastic labeling. Different dimension labels can be activated at different times. The dimension label provides information about the order in which targets, groups of targets, and/or samples were stochastically barcoded. For example, a population of cells can be stochastically barcoded at the G0 phase of the cell cycle. The cells can be pulsed again with stochastic barcodes at the G1 phase of the cell cycle. The cells can be pulsed again with stochastic barcodes at the S phase of the cell cycle, and so on. Stochastic barcodes at each pulse (e.g., each phase of the cell cycle), can comprise different dimension labels. In this way, the dimension label provides information about which targets were labelled at which phase of the cell cycle. Dimension labels can interrogate many different biological times. Exemplary biological times can include, but are not limited to, the cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a population of cells) can be stochastically labeled before and/or after treatment with a drug and/or therapy. The changes in the number of copies of distinct targets can be indicative of the sample's response to the drug and/or therapy.

In some embodiments, a dimension label is activatable. An activatable dimension label can be activated, for example, at a specific timepoint. The activatable dimension label can be constitutively activated (e.g., not turned off). The activatable dimension label can be reversibly activated (e.g., the activatable dimension label can be turned on and turned off). The dimension label can be reversibly activatable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. The dimension label can be reversibly activatable 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. For example, the dimension label can be activated with fluorescence, light, a chemical event (e.g., cleavage, ligation of another molecule, addition of modifications (e.g., pegylated, sumoylated, acetylated, methylated, deacetylated, demethylated), a photochemical event (e.g., photocaging), and introduction of a non-natural nucleotide.

The dimension label can be identical for all stochastic barcodes attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100% of stochastic barcodes on the same solid support comprise the same dimension label. In some embodiments, at least 60% of stochastic barcodes on the same solid support comprise the same dimension label. In some embodiments, at least 95% of stochastic barcodes on the same solid support comprise the same dimension label.

There can be as many as 10⁶ or more unique dimension label sequences represented in a plurality of solid supports (e.g., beads). A dimension label can, for example, be or be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. A dimension label can be at most about 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or fewer or more nucleotides in length. A dimension label can, for example, is about 5 to about 200 nucleotides, or about 10 to about 150 nucleotides in length. In some embodiments, a dimension label is from about 20 to about 125 nucleotides in length.

A stochastic barcode can comprise a spatial label. A spatial label can comprise a nucleic acid sequence that provides information about the spatial orientation of a target molecule which is associated with the stochastic barcode. A spatial label can be associated with a coordinate in a sample. The coordinate can be a fixed coordinate. For example a coordinate can be fixed in reference to a substrate. A spatial label can be in reference to a two or three-dimensional grid. A coordinate can be fixed in reference to a landmark. The landmark can be identifiable in space. A landmark can be a structure which can be imaged. A landmark can be a biological structure, for example an anatomical landmark. A landmark can be a cellular landmark, for instance an organelle. A landmark can be a non-natural landmark such as a structure with an identifiable identifier such as a color code, bar code, magnetic property, fluorescents, radioactivity, or a unique size or shape. A spatial label can be associated with a physical partition (e.g. a well, a container, or a droplet). In some instances, multiple spatial labels are used together to encode one or more positions in space.

The spatial label can be identical for all stochastic barcodes attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100% of stochastic barcodes on the same solid support comprise the same spatial label. In some embodiments, at least 60% of stochastic barcodes on the same solid support comprise the same spatial label. In some embodiments, at least 95% of stochastic barcodes on the same solid support comprise the same spatial label.

There can be as many as 10⁶ or more unique spatial label sequences represented in a plurality of solid supports (e.g., beads). A spatial label can be, or be at least about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, a spatial label is most about 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 nucleotides in length. A spatial label can be, for example, from about 5 to about 200 nucleotides in length. A spatial label can be, for example, from about 10 to about 150 nucleotides in length. A spatial label can be from about 20 to about 125 nucleotides in length.

Stochastic barcodes can comprise a cellular label. A cellular label can comprise a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell. In some embodiments, the cellular label is identical for all stochastic barcodes attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100% of stochastic barcodes on the same solid support comprise the same cellular label. In some embodiments, at least 60% of stochastic barcodes on the same solid support comprise the same cellular label. In some embodiment, at least 95% of stochastic barcodes on the same solid support comprise the same cellular label.

There can be as many as 10⁶ or more unique cellular label sequences represented in a plurality of solid supports (e.g., beads). A cellular label may be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. A cellular label can be, or be at most about, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or fewer or more nucleotides in length. A cellular label can be, for example, from about 5 to about 200 nucleotides in length. A cellular label can be, for example, from about 10 to about 150 nucleotides in length. A cellular label can be, for example, from about 20 to about 125 nucleotides in length.

Stochastic barcodes can comprise a molecular label. A molecular label can comprise a nucleic acid sequence that provides identifying information for the specific type of target nucleic acid species hybridized to the stochastic barcode. A molecular label can comprise a nucleic acid sequence that provides a counter for the specific occurrence of the target nucleic acid species hybridized to the stochastic barcode (e.g., target-binding region). In some embodiments, a diverse set of molecular labels are attached to a given solid support (e.g., bead). In some embodiments, there can be as many as 10⁶ or more unique molecular label sequences attached to a given solid support (e.g., bead). In some embodiments, there can be as many as 10⁵ or more unique molecular label sequences attached to a given solid support (e.g., bead). In some embodiments, there can be as many as 10⁴ or more unique molecular label sequences attached to a given solid support (e.g., bead). In some embodiments, there can be as many as 10³ or more unique molecular label sequences attached to a given solid support (e.g., bead). In some embodiments, there can be as many as 10² or more unique molecular label sequences attached to a given solid support (e.g., bead). A molecular label can be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. A molecular label can be at most about 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or fewer nucleotides in length.

Stochastic barcodes can comprise a target binding region. In some embodiments, the target binding regions comprise a nucleic acid sequence that hybridizes specifically to a target (e.g., target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), for example to a specific gene sequence. In some embodiments, a target binding region comprise a nucleic acid sequence that may attach (e.g., hybridize) to a specific location of a specific target nucleic acid. In some embodiments, the target binding region comprise a nucleic acid sequence that is capable of specific hybridization to a restriction site overhang (e.g. an EcoRI sticky-end overhang). The stochastic barcode may then ligate to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang.

A stochastic barcode can comprise a target-binding region. A target-binding region can hybridize with a target of interest. For example, a target-binding region can comprise an oligo dT which can hybridize with mRNAs comprising poly-adenylated ends. A target-binding region can be gene-specific. For example, a target-binding region can be configured to hybridize to a specific region of a target. A target-binding region can be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30 or more nucleotides in length. A target-binding region can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30 or more nucleotides in length. A target-binding region can be from 5-30 nucleotides in length. When a stochastic barcode comprises a gene-specific target-binding region, the stochastic barcode can be referred to as a gene-specific stochastic barcode.

A target binding region can comprise a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence can refer to a sequence that may bind to multiple target nucleic acids, independent of the specific sequence of the target nucleic acid. For example, target binding region can comprise a random multimer sequence, or an oligo-dT sequence that hybridizes to the poly-A tail on mRNA molecules. A random multimer sequence can be, for example, a random dimer, trimer, quatramer, pentamer, hexamer, septamer, octamer, nonamer, decamer, or higher multimer sequence of any length. In some embodiments, the target binding region is the same for all stochastic barcodes attached to a given bead. In some embodiments, the target binding regions for the plurality of stochastic barcodes attached to a given bead comprise two or more different target binding sequences. A target binding region can be, or be at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, a target binding region is at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.

A stochastic barcode can comprise an orientation property which can be used to orient (e.g., align) the stochastic barcodes. A stochastic barcode can comprise a moiety for isoelectric focusing. Different stochastic barcodes can comprise different isoelectric focusing points. When these stochastic barcodes are introduced to a sample, the sample can undergo isoelectric focusing in order to orient the stochastic barcodes into a known way. In this way, the orientation property can be used to develop a known map of stochastic barcodes in a sample. Exemplary orientation properties include, but are not limited to, electrophoretic mobility (e.g., based on size of the stochastic barcode), isoelectric point, spin, conductivity, and/or self-assembly. For example, stochastic barcodes can comprise an orientation property of self-assembly, can self-assemble into a specific orientation (e.g., nucleic acid nanostructure) upon activation.

A stochastic barcode can comprise an affinity property. A spatial label can comprise an affinity property. An affinity property can be included in a chemical and/or biological moiety that can facilitate binding of the stochastic barcode to another entity (e.g., cell receptor). For example, an affinity property can comprise an antibody. An antibody can be specific for a specific moiety (e.g., receptor) on a sample. An antibody can guide the stochastic barcode to a specific cell type or molecule. Targets at and/or near the specific cell type or molecule can be stochastically labeled. An affinity property can also provide spatial information in addition to the nucleotide sequence of the spatial label because the antibody can guide the stochastic barcode to a specific location. An antibody can be a therapeutic antibody. An antibody can be a monoclonal antibody. An antibody can be a polyclonal antibody. An antibody can be humanized. An antibody can be chimeric. An antibody can be a naked antibody. An antibody can be a fusion antibody.

An antibody, can be a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, like an antibody fragment.

An antibody can be an antibody fragment. An antibody fragment can be a portion of an antibody such as F(ab′)2, Fab′, Fab, Fv, sFv and the like. An antibody fragment can bind with the same antigen that is recognized by the full-length antibody. An antibody fragment can include isolated fragments consisting of the variable regions of antibodies, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). Exemplary antibodies can include, but are not limited to, antibodies for antibodies for cancer cells, antibodies for viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

The cellular label and/or any label of the disclosure can further comprise a unique set of nucleic acid sub-sequences of defined length, e.g. 7 nucleotides each (equivalent to the number of bits used in some Hamming error correction codes), which are designed to provide error correction capability. Hamming codes, like other error-correcting codes, are based on the principle of redundancy and can be constructed by adding redundant parity bits to data that is to be transmitted over a noisy medium. Such error-correcting codes can encode sample identifiers with redundant parity bits, and “transmit” these sample identifiers as codewords. A Hamming code can refer an arithmetic process that identifies unique binary codes based upon inherent redundancy that are capable of correcting single bit errors. For example, a Hamming code can be matched with a nucleic acid barcode in order to screen for single nucleotide errors occurring during nucleic acid amplification. The identification of a single nucleotide error by using a Hamming code, thereby can allow for the correction of the nucleic acid barcode.

Hamming codes can be represented by a subset of the possible codewords that are chosen from the center of multidimensional spheres (i.e., for example, hyperspheres) in a binary subspace. Single bit errors may fall within hyperspheres associated with a specific codeword and can thus be corrected. On the other hand, double bit errors that do not associate with a specific codeword can be detected, but not corrected. Consider a first hypersphere centered at coordinates (0, 0, 0) (i.e., for example, using an x-y-z coordinate system), wherein any single-bit error can be corrected by falling within a radius of 1 from the center coordinates; i.e., for example, single bit errors having the coordinates of (0, 0, 0); (0, 1, 0); (0, 0, 1); (1, 0, 0), or (1, 1, 0). Likewise, a second hypersphere may be constructed wherein single-bit errors can be corrected by falling within a radius of 1 of its center coordinates (1, 1, 1) (i.e., for example, (1,1,1); (1, 0, 1); (0, 1, 0); or (0, 1, 1).

In some embodiments, the length of the nucleic acid sub-sequences used for creating error correction codes can vary, for example, they can be at least 3 nucleotides, at least 7 nucleotides, at least 15 nucleotides, or at least 31 nucleotides in length. In some embodiments, nucleic acid sub-sequences of other lengths can be used for creating error correction codes.

When a stochastic barcode comprises more than one of a type of label (e.g., more than one cellular label or more than one molecular label), the labels may be interspersed with a linker label sequence. A linker label sequence can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. A linker label sequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. In some instances, a linker label sequence is 12 nucleotides in length. A linker label sequence can be used to facilitate the synthesis of the stochastic barcode. The linker label can comprise an error-correcting (e.g., Hamming) code.

Combinatorial Barcodes

A large number of different combinatorial barcodes can be used to label a large number of samples, such as single cells or nucleic acid fragments, for parallel analysis such as sequencing. As disclosed herein, a set of large number of combinatorial barcode can be generated from a relatively small number of barcode subunit sequences wherein the barcode subunit sequences are connected to each other by various combinations. In this way, the number of different combinatorial barcodes generated can be increased significantly through relatively small increases in the number of different barcode subunit sequences, the number of barcode subunit sequences connected to each other in a combinatorial barcode, or both.

Some embodiments disclosed herein provide compositions comprising a set of combinatorial barcodes. A combinatorial barcode can comprise two or more barcode subunit sequences that are connected to each other through a linker sequence, a target nucleotide sequence, or both. Two or more barcode subunit sequences can, for example, form a combinatorial barcode by connecting with each other through hybridization of one or more linker sequences followed by extension. For example, a linker sequence linked to a barcode subunit sequence may hybridize to a linker sequence linked to another barcode subunit sequence, and use the other barcode subunit sequence as a template in an extension reaction to incorporate the other barcode subunit sequence, and so on. In some embodiments, two barcode subunit sequences can both hybridize to a target nucleotide sequence, followed by two extension reactions to incorporate the two barcode subunit sequences to the target nucleotide sequence.

In some embodiments, the number of barcode subunit sequences in a combinatorial barcode, and thus the length of the combinatorial barcode, can be affected by the design of the linker sequences. For example, to generate a combinatorial barcode having m barcode subunit sequences, m−1 or m−2 linker sequences may be used, wherein m is an integer≥2. If n unique barcode subunit sequences are used to generate a combinatorial barcode having m barcode subunit sequences, a maximum number of n^(m) unique combinatorial barcodes may be generated. In some embodiments, the combinatorial barcode can comprise an oligonucleotide comprising the formula: barcode subunit sequences-linkers-barcode subunit sequence_(b)-linker₂- . . . barcode subunit sequence_(c)-linker_(m-1)-barcode subunit sequence_(d). In some embodiments, the combinatorial barcode may comprise a first oligonucleotide comprising the formula barcode subunit sequence_(a)-linker₁-barcode subunit sequence_(b)-linker₂- . . . -barcode subunit sequence; and a second oligonucleotide comprising barcode subunit sequence_(d)- . . . -linker₃-barcode subunit sequence_(e)-linker_(m-2)-barcode subunit sequence_(f). The first oligonucleotide and the second oligonucleotide may be connected to each other by a target nucleotide sequence. In some embodiments, each one of the barcode subunit sequence_(a), barcode subunit sequence_(b), barcode subunit sequence_(c), . . . in a combinatorial barcode is selected from a set of n unique barcode subunit sequences. In some embodiments, some or all of the barcode subunit sequence_(a), barcode subunit sequence_(b), barcode subunit sequence_(c), . . . in a combinatorial barcode may be the same. In some embodiments, some or all of the barcode subunit sequences, barcode subunit sequence_(b), barcode subunit sequence, . . . in a combinatorial barcode may be different.

In some embodiments, a set of combinatorial barcodes comprise at least 1,000, at least 10,000, at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, or more unique combinatorial barcodes.

A combinatorial barcode can comprise one or more stochastic barcodes disclosed herein. For example, a combinatorial barcode can comprise one or more universal labels, cellular labels, molecular labels, sample labels, plate labels, spatial labels, pre-spatial labels, or any combination thereof. In some embodiments, a stochastic barcode, such as a cellular label, a sample label, a spatial label, etc., can comprise two or more barcode subunit sequences, for example, at least 2, at least 3, at least 4, at least 5, or more barcode subunit sequences.

In some embodiments, the combinatorial barcodes may be immobilized on a solid support, such as a bead or a microwell in a microwell array. As shown in FIG. 8 , a solid support, such as a bead or a microparticle, may be coated with a first plurality of combinatorial barcodes. In some embodiments, the combinatorial barcodes can comprise one or more of a universal sequence (US), a cellular label, a linker, a molecular label, a target-specific region such as an oligo(dT) sequence. In some embodiments, the solid support may comprise a second plurality of combinatorial barcodes that has the same structure (but not necessarily the same sequence) as the first plurality of combinatorial barcodes, except for having a spatial primer instead of a target-specific region. In some embodiments, second plurality of combinatorial barcodes is less in number, for example, 1/10, 1/100, 1/1,000 the number, of the first plurality of combinatorial barcodes. In some embodiments, the second plurality of combinatorial barcodes is used for introducing a spatial label via universal PCR. The second plurality of combinatorial barcodes may be added by printing or dispensing into each well. In some embodiments, the second plurality of combinatorial barcode may be delivered by a second bead into the same well with a first bead.

Component Barcodes

As disclosed herein, two or more component barcodes each comprising a barcode subunit sequence can be connected to each other via one or more linker sequences to generate a combinatorial barcode. For example, each of the component barcode can comprise a barcode subunit sequence and one or more linker sequences or the complement thereof, and two or more component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes. A component barcode can comprise a barcode subunit sequence, a linker sequence, a target-specific region, or any combination thereof. For example, a component barcode may comprise one of the following configurations: a) barcode subunit sequence-linker sequence (i.e., the component barcode comprises both a barcode unit sequence and a linker sequence, where the barcode subunit sequence is located at the 5′ portion of the component barcode as relative to the linker sequence); b) complement of linker sequence-barcode subunit sequence-linker sequence; c) complement of linker sequence-barcode subunit sequence; d) barcode subunit sequence-complement of linker sequence; e) linker sequence-barcode subunit sequence-complement of linker sequence; or f) linker sequence-barcode subunit sequence. In some embodiments, a barcode subunit sequence and a linker sequence can be linked not directly by a chemical bond. For example, a barcode subunit sequence and a linker sequence can be linked via a chemical or biological moiety.

A component barcode, or combinatorial barcode reagent, can comprise a subunit code section which comprises a barcode subunit sequence. A combinatorial barcode reagent can comprise 1, 2, 3, 4 or 5 or more subunit code sections. A combinatorial barcode reagent can comprise at least, or at most, 1, 2, 3, 4 or 5 subunit code sections. In some instances, a combinatorial barcode reagent has 1 subunit code section. A subunit code section can comprise a subunit code sequence. A subunit code sequence, or a barcode subunit sequence, which are used interchangeably, can refer to a unique sequence of nucleotides in the combinatorial barcode reagent. A subunit code section can be, or be at least, 5, 10, 15, 20, 25, 30, 35, 40, or 45 or more nucleotides in length. A subunit code section can be at most 5, 10, 15, 20, 25, 30, 35, 40, or 45 nucleotides in length. In some instances, a subunit code section is 6 nucleotides in length.

In some embodiments, each position of the subunit code section have an option of 4 nucleotides (e.g., A, T, C, and G). If each subunit code section have a length of n nucleotides, where n is a positive integer, the number of possible unique subunit code sequences for a subunit code section can be 4′ For example, if a subunit code section is 6 nucleotides in length, and each position has an option of 4 nucleotides, the total number of possible unique subunit code sequences can be 4,096 codes.

A subset of possible subunit code sequences can be used as combinatorial barcode reagents of the disclosure. The subset of possible subunit code sequences can be selected based on their error-correcting properties. For example, the subset of possible subunit code sequences can have sequences that are far enough apart (e.g., have different enough sequences, distance) such that the subunit code sequences can correct for base errors (e.g., 1 or 2 base errors). In another example, the subset of subunit code sequences can be of a length and number that incorporate error-correcting barcodes of the disclosure (e.g., Hamming codes) into the sequences.

The number of subunit code sequences in a subset can be, or be at least, 5, 10, 15, 20, 25, 30, 35, 40, or 45 or more. In some embodiments, the number of subunit code sequences in a subset is at most 5, 10, 15, 20, 25, 30, 35, 40, or 45. In some embodiments, the number of subunit code sequences in a subset is 24. Subunit code sequences can be different from each other. Subunit code sequences can differ by, or by at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. In some embodiments, subunit code sequences can differ by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Subunit code sequences may be a non-cross hybridizing set of sequences. Subunit code sequences may be error-correcting.

A combinatorial barcode reagent can comprise one or more linkers. A linker can be used to link combinatorial barcode reagents together (e.g., concatenate combinatorial barcode reagents together). A linker can be configured to hybridize to a linker of another combinatorial barcode reagent (e.g., thereby concatenating the combinatorial barcode reagents). A linker can be 3′ of the subunit code of the combinatorial barcode reagent. A linker can be 5′ of the subunit code of the combinatorial barcode reagent. A linker can be both 3′ and 5′ of the subunit code of the combinatorial barcode reagent. A linker can be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. In some embodiments, a linker can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. In some embodiments, a linker sequence of a first combinatorial barcode reagent can hybridize to a linker sequence of a second combinatorial barcode reagent. In some embodiments, a linker sequence of a first combinatorial barcode reagent can be the complement of a linker sequence of a second combinatorial barcode reagent. In some embodiments, a linker sequence of a first combinatorial barcode reagent can hybridize to a linker sequence of a second combinatorial barcode reagent with, or with at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches. In some embodiments, a linker sequence of a first combinatorial barcode reagent can hybridize to a linker sequence of a second combinatorial barcode reagent with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches.

In some embodiments, a combinatorial barcode reagent comprise a target-specific region. A target specific region can associate with (e.g., hybridize) with a target polynucleotide of the disclosure (e.g., from a single cell). A target-specific region can comprise an oligo-dT, a gene-specific sequence, or a random multimer sequence. A target-specific region can hybridize to a sense strand of a double-stranded target polynucleotide of the disclosure. A target-specific region can hybridize to an anti-sense strand of a double-stranded target polynucleotide of the disclosure. A target-specific region can be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. In some embodiments, a target-specific region can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

In some embodiments, a combinatorial barcode reagent comprise one or more cellular labels, molecular labels, universal labels, target-binding regions, or any labels of the disclosure, or any combination thereof.

Combinatorial barcode reagents can be comprised of any type of nucleic acid (e.g., PNA, LNA). Combinatorial barcode reagents can be attached to a solid or semi-support (e.g., a bead, a gel particle, an antibody, a hydrogel, agarose). Combinatorial barcode reagents can be immobilized on a substrate of the disclosure (e.g., an array). Combinatorial barcode reagents can be incorporated into a biological package, such as a virus, a liposome, a microsphere, etc. Combinatorial reagents can comprise moieties. Moieties can act as identifiers (e.g., fluorescent moiety, radioactive moiety).

Sets of Component Barcodes

Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes. The number of unique combinatorial barcode reagents in a set can be n×m, where n is the number of subunit code sequences (n) and m is the number of subunit code sections in a combinatorial barcode (m), and where n and m are positive integers. For example, when the subset of subunit code sequences is 24 and there are 4 subunit code sections in a combinatorial barcode, there can be 96 total unique combinatorial barcode reagents. The maximum number of the set of unique combinatorial barcodes that can be generated using the n×m unique component barcode is n^(m), where n is the number of subunit code sequences and m is the number of subunit code sections in a combinatorial barcode. For example, if there are 24 subunit code sequences and 4 subunit code sections in a combinatorial barcode, a maximum set of 331,776 unique combinatorial barcodes can be generated from the set of 96 unique combinatorial barcode reagents.

The number of subunit code sections in a combinatorial barcode can be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The number of subunit code sections in a combinatorial barcode can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the number of subunit code sections in a combinatorial barcode is 4 (as shown in FIG. 1 ).

Some combinatorial barcode reagents of the set of combinatorial barcode reagents can comprise a target-specific region. The number of combinatorial barcode reagents in a set that comprise a target-specific region can be, or be at least, 1, 2, 3, 4, or 5 or more. In some embodiments, the number of combinatorial barcode reagents in a set that comprise a target-specific region can be at most 1, 2, 3, 4, or 5. As shown in FIGS. 2A-C, in some embodiments, the number of combinatorial barcode reagents in a set that comprise a target-specific region is at least 2. In some instances, the number of combinatorial barcode reagents in a set that comprise a target-specific region is 2 (FIGS. 2A-B). In some embodiments, the number of combinatorial barcode reagents in a set that comprise a target-specific region is at least 1. In some instances, the number of combinatorial barcode reagents in a set that comprise a target-specific region is 1 (FIG. 2C).

A set of combinatorial barcode reagents can comprise some combinatorial barcode reagents comprising a target-specific region and some combinatorial barcode reagents not comprising a target-specific region. In some instances, a set of combinatorial barcode reagents comprises one combinatorial barcode reagent with a target-specific region, and the rest of the combinatorial barcode reagents may not have a target-specific region (e.g., may comprise a subunit code section/sequence and one or more linkers). In some instances, a set of combinatorial barcode reagents comprises two combinatorial barcode reagents with a target-specific region, and the rest of the combinatorial barcode reagents may not have a target-specific region (e.g., may comprise a subunit code section/sequence and one or more linkers).

The number of combinatorial barcode reagents in a set that may not have a target-specific region can be, or be least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, the number of combinatorial barcode reagents in a set that may not have a target-specific region can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

A set of combinatorial barcode reagents can encode for a plurality of combinatorial barcodes. A combinatorial barcode can comprise the total sequence of the combinatorial barcodes reagents from the set (i.e., for a given target polynucleotide). A combinatorial barcode can be the total sequence of concatenated combinatorial barcode reagents (e.g., linked together through linkers) for a given target polynucleotide. A combinatorial barcode can include the linker sequences of the combinatorial barcode reagents. A combinatorial barcode can exclude the linker sequences of the combinatorial barcode reagents (e.g., only include the subunit code sequences). A combinatorial barcode can be the total sequence of the sequences of the subunit code sections of the combinatorial barcode reagents, for a given target polynucleotide.

In some embodiments, a combinatorial barcode can refer to (e.g., be formed when) two or more combinatorial barcode reagents hybridized to each other through one or more linker sequences and/or hybridized to a target polynucleotide through one or more target-specific regions. In some embodiments, a combinatorial barcode can refer to two or more combinatorial barcode reagents incorporated into a single polynucleotide with or without a target polynucleotide through extension, reverse transcription, and/or amplification.

In some embodiments, a combinatorial barcode can be bipartite. A portion of a combinatorial barcode can be connected to the 5′ end of a target polynucleotide. A portion of a combinatorial barcode can be connected to the 3′ end of the target polynucleotide. The entire combinatorial barcode can be connected to the 5′ end of a target polynucleotide. The entire combinatorial barcode can be connected to the 3′ end of a target polynucleotide.

A combination of combinatorial barcode reagents can make a combinatorial barcode. The combinatorial barcode can comprise one or more cellular labels, molecular labels, universal labels, target-binding regions, any labels of the disclosure, or any combination thereof. In some instances, a combinatorial barcode reagent may not comprise a label. For example, if a combinatorial barcode is made up of 4 combinatorial barcode reagents, 1 combinatorial barcode reagent may not comprise a label, and 3 combinatorial barcode reagents of the combinatorial barcode may comprise any label disclosed herein. A combinatorial barcode reagent can comprise a stochastic barcode of the disclosure.

The set of combinatorial barcode reagents can be random. For example, all or some of the linker sequences in a combinatorial barcode may be the same. Therefore, the component barcodes can be linked in any order. The set of combinatorial barcode reagents can be non-random. For example, all of the linker sequences in a combinatorial barcode may be different. Therefore, the order of the component barcodes can be linked in a specific order.

In some embodiments, a combinatorial barcode reagent is attached to any solid or semi-solid support disclosed herein. For example, the combinatorial barcode reagents can be attached to a combination of a bead and a gel particle, or a first combinatorial barcode reagent in solution combined with a second combinatorial barcode reagent attached on a bead, or a first combinatorial barcode reagent immobilized on a substrate within a microwell and a second combinatorial barcode reagent in solution or present on a solid particle within the microwell. In some embodiments, a combinatorial barcode reagent is embedded within hydrogels or similar materials that can act as a sponge-like scaffold for the purpose of fixed position localization of biological samples and nucleic acids.

Solid Supports

The combinatorial barcode reagents and/or combinatorial barcodes disclosed herein can be attached to a solid support (e.g., bead, substrate). The combinatorial barcode reagents and/or combinatorial barcodes disclosed herein can be located in a solid support (e.g., in a microwell of an array). As used herein, the terms “tethered”, “attached”, and “immobilized” are used interchangeably, and refer to covalent or non-covalent means for attaching stochastic barcodes to a solid support. Any of a variety of different solid supports may be used as solid supports for attaching pre-synthesized combinatorial barcode reagents or for in situ solid-phase synthesis of combinatorial barcode reagents.

In some instances, a solid support is a bead. A bead can encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently). A bead can comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. A bead can be non-spherical in shape.

Beads can comprise a variety of materials including, but not limited to, paramagnetic materials (e.g. magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g. ferrite (Fe₃O₄; magnetite) nanoparticles), ferromagnetic materials (e.g. iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogel, polymer, cellulose, nylon, and any combination thereof.

The diameter of the beads can be, or be at least about, 5 μm, 10 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm. The diameter of the beads can be at most about 5 μm, 10 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm. The diameter of the bead can be related to the diameter of the wells of the substrate. For example, the diameter of the bead can be, or be at least, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% longer or shorter than the diameter of the well. In some embodiments, the diameter of the bead can be at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% longer or shorter than the diameter of the well. The diameter of the bead may be related to the diameter of a cell (e.g., a single cell entrapped by the a well of the substrate). The diameter of the bead can be, or be at least, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300% or more longer or shorter than the diameter of the cell. In some embodiments, the diameter of the bead can be at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300% or more longer or shorter than the diameter of the cell.

A bead can be attached to and/or embedded in a substrate of the disclosure. A bead can be attached to and/or embedded in a gel, hydrogel, polymer and/or matrix. The spatial position of a bead within a substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using the spatial label present on the stochastic barcode on the bead which can serve as a location address.

Examples of beads can include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugated beads (e.g., anti-immunoglobulin microbead), protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligodT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and BcMag™ Carboxy-Terminated Magnetic Beads.

A bead can be associated with (e.g. impregnated with) quantum dots or fluorescent dyes to make it fluorescent in one fluorescence optical channel or multiple optical channels. A bead can be associated with iron oxide or chromium oxide to make it paramagnetic or ferromagnetic. Beads can be identifiable. A bead can be imaged using a camera. A bead can have a detectable code associated with the bead. For example, a bead can comprise an RFID tag. A bead can comprise any detectable tag (e.g., UPC code, electronic barcode, etched identifier). A bead can change size, for example due to swelling in an organic or inorganic solution. A bead can be hydrophobic or hydrophilic. A bead can be biocompatible.

A solid support (e.g., bead) can be visualized. The solid support can comprise a visualizing tag (e.g., fluorescent dye). A solid support (e.g., bead) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging the solid supports (e.g., beads).

A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as “functionalized” when it includes a linker, a scaffold, a building block, or other reactive moiety attached thereto, whereas a solid support may be “nonfunctionalized” when it lack such a reactive moiety attached thereto. The solid support can be employed free in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.

The solid support can comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. A solid support can take the form of resins, gels, microspheres, or other geometric configurations. A solid support can comprise silica chips, microparticles, nanoparticles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microwell plates, slides, plastic materials including multiwell plates or membranes (e.g., formed of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g., silicon wafers), wafers with pits with or without filter bottoms.

The solid support can comprise a polymer matrix (e.g., gel, hydrogel). The polymer matrix may be able to permeate intracellular space (e.g., around organelles). The polymer matrix may able to be pumped throughout the circulatory system.

A solid support can be a biological molecule. For example a solid support can be a nucleic acid, a protein, an antibody, a histone, a cellular compartment, a lipid, a carbohydrate, and the like. Solid supports that are biological molecules can be amplified, translated, transcribed, degraded, and/or modified (e.g., pegylated, sumoylated, acetylated, methylated). A solid support that is a biological molecule can provide spatial and time information in addition to the spatial label that is attached to the biological molecule. For example, a biological molecule can comprise a first confirmation when unmodified, but can change to a second confirmation when modified. The different conformations can expose stochastic barcodes of the disclosure to targets. For example, a biological molecule can comprise stochastic barcodes that are unaccessible due to folding of the biological molecule. Upon modification of the biological molecule (e.g., acetylation), the biological molecule can change conformation to expose the stochastic labels. The timing of the modification can provide another time dimension to the method of stochastic barcoding of the disclosure.

In some embodiments, the biological molecule comprising combinatorial barcode reagents of the disclosure can be located in the cytoplasm of a cell. Upon activation, the biological molecule can move to the nucleus, whereupon stochastic barcoding can take place. In this way, modification of the biological molecule can encode additional space-time information for the targets identified by the stochastic barcodes.

A dimension label can provide information about space-time of a biological event (e.g., cell division). For example, a dimension label can be added to a first cell, the first cell can divide generating a second daughter cell, the second daughter cell can comprise all, some or none of the dimension labels. The dimension labels can be activated in the original cell and the daughter cell. In this way, the dimension label can provide information about time of combinatorial barcoding in distinct spaces.

Substrates

A substrate can refer to a type of solid support. A substrate can refer to a solid support that can comprise combinatorial barcode reagents of the disclosure. A substrate can comprise a plurality of microwells. A microwell can comprise a small reaction chamber of defined volume. A microwell can entrap one or more cells. A microwell can entrap only one cell. A microwell can entrap one or more solid supports. A microwell can entrap only one solid support. In some instances, a microwell entraps a single cell and a single solid support (e.g., bead). A microwell can comprise combinatorial barcode reagents of the disclosure.

The microwells of the array can be fabricated in a variety of shapes and sizes. Well geometries can include, but are not limited to, cylindrical, conical, hemispherical, rectangular, or polyhedral (e.g., three dimensional geometries comprised of several planar faces, for example, hexagonal columns, octagonal columns, inverted triangular pyramids, inverted square pyramids, inverted pentagonal pyramids, inverted hexagonal pyramids, or inverted truncated pyramids). The microwells can comprise a shape that combines two or more of these geometries. For example, a microwell can be partly cylindrical, with the remainder having the shape of an inverted cone. A microwell can include two side-by-side cylinders, one of larger diameter (e.g. that corresponds roughly to the diameter of the beads) than the other (e.g. that corresponds roughly to the diameter of the cells), that are connected by a vertical channel (that is, parallel to the cylinder axes) that extends the full length (depth) of the cylinders. The opening of the microwell can be at the upper surface of the substrate. The opening of the microwell can be at the lower surface of the substrate. The closed end (or bottom) of the microwell can be flat. The closed end (or bottom) of the microwell can have a curved surface (e.g., convex or concave). The shape and/or size of the microwell can be determined based on the types of cells or solid supports to be trapped within the microwells.

The portion of the substrate between the wells can have a topology. For example, the portion of the substrate between the wells can be rounded. The portion of the substrate between the wells can be pointed. The spacing portion of the substrate between the wells can be flat. The portion of the substrate between the wells may not be flat. In some instances, the portion of the substrate between wells is rounded. In other words, the portion of the substrate that does not comprise a well can have a curved surface. The curved surface can be fabricated such that the highest point (e.g., apex) of the curved surface may be at the furthest point between the edges of two or more wells (e.g., equidistant from the wells). The curved surface can be fabricated such that the start of the curved surface is at the edge of a first microwell and creates a parabola that ends at the end of a second microwell. This parabola can be extended in 2 dimensions to capture microwells nearby on the hexagonal grid of wells. The curved surface can be fabricated such that the surface between the wells is higher and/or curved than the plane of the opening of the well. The height of the curved surface can be, or be at least, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 or more micrometers. In some embodiments, the height of the curved surface can be at most 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 or more micrometers.

Microwell dimensions can be characterized in terms of the diameter and depth of the well. As used herein, the diameter of the microwell refers to the largest circle that can be inscribed within the planar cross-section of the microwell geometry. The diameter of the microwells can range from about 1-fold to about 10-fold the diameter of the cells or solid supports to be trapped within the microwells. The microwell diameter can be, or be at least, 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold the diameter of the cells or solid supports to be trapped within the microwells. In some embodiments, the microwell diameter can be at most 10-fold, at most 5-fold, at most 4-fold, at most 3-fold, at most 2-fold, at most 1.5-fold, or at most 1-fold the diameter of the cells or solid supports to be trapped within the microwells. The microwell diameter can be about 2.5-fold the diameter of the cells or solid supports to be trapped within the microwells.

The diameter of the microwells can be specified in terms of absolute dimensions. The diameter of the microwells can range from about 5 to about 60 micrometers. The microwell diameter can be, or be at least, 5 micrometers, at least 10 micrometers, at least 15 micrometers, at least 20 micrometers, at least 25 micrometers, at least 30 micrometers, at least 35 micrometers, at least 40 micrometers, at least 45 micrometers, at least 50 micrometers, or at least 60 micrometers. The microwell diameter can be at most 60 micrometers, at most 50 micrometers, at most 45 micrometers, at most 40 micrometers, at most 35 micrometers, at most 30 micrometers, at most 25 micrometers, at most 20 micrometers, at most 15 micrometers, at most 10 micrometers, or at most 5 micrometers. The microwell diameter can be about 30 micrometers.

The microwell depth may be chosen to provide efficient trapping of cells and solid supports. The microwell depth may be chosen to provide efficient exchange of assay buffers and other reagents contained within the wells. The ratio of diameter to height (i.e. aspect ratio) may be chosen such that once a cell and solid support settle inside a microwell, they will not be displaced by fluid motion above the microwell. The dimensions of the microwell may be chosen such that the microwell has sufficient space to accommodate a solid support and a cell of various sizes without being dislodged by fluid motion above the microwell. The depth of the microwells can range from about 1-fold to about 10-fold the diameter of the cells or solid supports to be trapped within the microwells. The microwell depth can be, or be at least, 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold the diameter of the cells or solid supports to be trapped within the microwells. The microwell depth can be at most 10-fold, at most 5-fold, at most 4-fold, at most 3-fold, at most 2-fold, at most 1.5-fold, or at most 1-fold the diameter of the cells or solid supports to be trapped within the microwells. The microwell depth can be about 2.5-fold the diameter of the cells or solid supports to be trapped within the microwells.

The depth of the microwells can be specified in terms of absolute dimensions. The depth of the microwells may range from about 10 to about 60 micrometers. The microwell depth can be, or be at least, 10 micrometers, at least 20 micrometers, at least 25 micrometers, at least 30 micrometers, at least 35 micrometers, at least 40 micrometers, at least 50 micrometers, or at least 60 micrometers. The microwell depth can be at most 60 micrometers, at most 50 micrometers, at most 40 micrometers, at most 35 micrometers, at most 30 micrometers, at most 25 micrometers, at most 20 micrometers, or at most 10 micrometers. The microwell depth can be about 30 micrometers.

The volume of the microwells used in the methods, devices, and systems of the present disclosure can range from about 200 micrometers³ to about 120,000 micrometers³. The microwell volume can be at least 200 micrometers³, at least 500 micrometers³, at least 1,000 micrometers³, at least 10,000 micrometers³, at least 25,000 micrometers³, at least 50,000 micrometers³, at least 100,000 micrometers³, or at least 120,000 micrometers³. The microwell volume can be at most 120,000 micrometers³, at most 100,000 micrometers³, at most 50,000 micrometers³, at most 25,000 micrometers³, at most 10,000 micrometers³, at most 1,000 micrometers³, at most 500 micrometers³, or at most 200 micrometers³. The microwell volume can be about 25,000 micrometers³. The microwell volume may fall within any range bounded by any of these values (e.g. from about 18,000 micrometers³ to about 30,000 micrometers³).

The volume of the microwell can be, or be at least, 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more nanoliters³. The volume of the microwell can be at most 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more nanoliters³. The volume of liquid that can fit in the microwell can be at least 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more nanoliters³. The volume of liquid that can fit in the microwell can be at most 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more nanoliters³. The volume of the microwell can be, or be at least, 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more picoliters³. The volume of the microwell can be at most 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more picoliters³. The volume of liquid that can fit in the microwell can be at least 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more picoliters³. The volume of liquid that can fit in the microwell can be at most 5, 10, 15, 20, 25, 30, 35 40, 45 or 50 or more picoliters³.

The volumes of the microwells used in the methods, devices, and systems of the present disclosure may be further characterized in terms of the variation in volume from one microwell to another. The coefficient of variation (expressed as a percentage) for microwell volume may range from about 1% to about 10%. The coefficient of variation for microwell volume may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%. The coefficient of variation for microwell volume may be at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%. The coefficient of variation for microwell volume may have any value within a range encompassed by these values, for example between about 1.5% and about 6.5%. In some embodiments, the coefficient of variation of microwell volume may be about 2.5%.

The ratio of the volume of the microwells to the surface area of the beads (or to the surface area of a solid support to which stochastic barcode oligonucleotides may be attached) used in the methods, devices, and systems of the present disclosure can range from about 2.5 to about 1,520 micrometers. The ratio can be at least 2.5, at least 5, at least 10, at least 100, at least 500, at least 750, at least 1,000, or at least 1,520. The ratio can be at most 1,520, at most 1,000, at most 750, at most 500, at most 100, at most 10, at most 5, or at most 2.5. The ratio can be about 67.5. The ratio of microwell volume to the surface area of the bead (or solid support used for immobilization) may fall within any range bounded by any of these values (e.g. from about 30 to about 120).

The wells of the microwell array can be arranged in a one dimensional, two dimensional, or three-dimensional array. In some embodiments, a three dimensional array can be achieved, for example, by stacking a series of two or more two dimensional arrays (that is, by stacking two or more substrates comprising microwell arrays).

The pattern and spacing between microwells can be chosen to optimize the efficiency of trapping a single cell and single solid support (e.g., bead) in each well, as well as to maximize the number of wells per unit area of the array. The microwells may be distributed according to a variety of random or non-random patterns. For example, they may be distributed entirely randomly across the surface of the array substrate, or they may be arranged in a square grid, rectangular grid, hexagonal grid, or the like. In some instances, the microwells are arranged hexagonally. The center-to-center distance (or spacing) between wells may vary from about 5 micrometers to about 75 micrometers. In some instances, the spacing between microwells is about 10 micrometers. In other embodiments, the spacing between wells is at least 5 micrometers, at least 10 micrometers, at least 15 micrometers, at least 20 micrometers, at least 25 micrometers, at least 30 micrometers, at least 35 micrometers, at least 40 micrometers, at least 45 micrometers, at least 50 micrometers, at least 55 micrometers, at least 60 micrometers, at least 65 micrometers, at least 70 micrometers, or at least 75 micrometers. The microwell spacing can be at most 75 micrometers, at most 70 micrometers, at most 65 micrometers, at most 60 micrometers, at most 55 micrometers, at most 50 micrometers, at most 45 micrometers, at most 40 micrometers, at most 35 micrometers, at most 30 micrometers, at most 25 micrometers, at most 20 micrometers, at most 15 micrometers, at most 10 micrometers, at most 5 micrometers. The microwell spacing can be about 55 micrometers. The microwell spacing may fall within any range bounded by any of these values (e.g. from about 18 micrometers to about 72 micrometers).

The microwell array may comprise surface features between the microwells that are designed to help guide cells and solid supports into the wells and/or prevent them from settling on the surfaces between wells. Examples of suitable surface features can include, but are not limited to, domed, ridged, or peaked surface features that encircle the wells or straddle the surface between wells.

The total number of wells in the microwell array can be determined by the pattern and spacing of the wells and the overall dimensions of the array. The number of microwells in the array can range from about 96 to about 5,000,000 or more. The number of microwells in the array can be at least 96, at least 384, at least 1,536, at least 5,000, at least 10,000, at least 25,000, at least 50,000, at least 75,000, at least 100,000, at least 500,000, at least 1,000,000, or at least 5,000,000. The number of microwells in the array can be at most 5,000,000, at most 1,000,000, at most 75,000, at most 50,000, at most 25,000, at most 10,000, at most 5,000, at most 1,536, at most 384, or at most 96 wells. The number of microwells in the array can be about 96, 384, and/or 1536. The number of microwells can be about 150,000. The number of microwells in the array may fall within any range bounded by any of these values (e.g. from about 100 to 325,000).

Microwell arrays may be fabricated using any of a number of fabrication techniques. Examples of fabrication methods that may be used include, but are not limited to, bulk micromachining techniques such as photolithography and wet chemical etching, plasma etching, or deep reactive ion etching; micro-molding and micro-embossing; laser micromachining; 3D printing or other direct write fabrication processes using curable materials; and similar techniques.

Microwell arrays can be fabricated from any of a number of substrate materials. The choice of material can depend on the choice of fabrication technique, and vice versa. Examples of suitable materials can include, but are not limited to, silicon, fused-silica, glass, polymers (e.g. agarose, gelatin, hydrogels, polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxy resins, thiol-ene based resins, metals or metal films (e.g. aluminum, stainless steel, copper, nickel, chromium, and titanium), and the like. In some instances, the microwell comprises optical adhesive. In some instances, the microwell is made out of optical adhesive. In some instances, the microwell array comprises and/or is made out of PDMS. In some instances, the microwell is made of plastic. A hydrophilic material can be desirable for fabrication of the microwell arrays (e.g. to enhance wettability and minimize non-specific binding of cells and other biological material). Hydrophobic materials that can be treated or coated (e.g. by oxygen plasma treatment, or grafting of a polyethylene oxide surface layer) can also be used. The use of porous, hydrophilic materials for the fabrication of the microwell array may be desirable in order to facilitate capillary wicking/venting of entrapped air bubbles in the device. The microwell array can be fabricated from a single material. The microwell array may comprise two or more different materials that have been bonded together or mechanically joined.

Microwell arrays can be fabricated using substrates of any of a variety of sizes and shapes. For example, the shape (or footprint) of the substrate within which microwells are fabricated may be square, rectangular, circular, or irregular in shape. The footprint of the microwell array substrate can be similar to that of a microtiter plate. The footprint of the microwell array substrate can be similar to that of standard microscope slides, e.g. about 75 mm long×25 mm wide (about 3″ long×1″ wide), or about 75 mm long×50 mm wide (about 3″ long×2″ wide). The thickness of the substrate within which the microwells are fabricated may range from about 0.1 mm thick to about 10 mm thick, or more. The thickness of the microwell array substrate may be at least 0.1 mm thick, at least 0.5 mm thick, at least 1 mm thick, at least 2 mm thick, at least 3 mm thick, at least 4 mm thick, at least 5 mm thick, at least 6 mm thick, at least 7 mm thick, at least 8 mm thick, at least 9 mm thick, or at least 10 mm thick. The thickness of the microwell array substrate may be at most 10 mm thick, at most 9 mm thick, at most 8 mm thick, at most 7 mm thick, at most 6 mm thick, at most 5 mm thick, at most 4 mm thick, at most 3 mm thick, at most 2 mm thick, at most 1 mm thick, at most 0.5 mm thick, or at most 0.1 mm thick. The thickness of the microwell array substrate can be about 1 mm thick. The thickness of the microwell array substrate may be any value within these ranges, for example, the thickness of the microwell array substrate may be between about 0.2 mm and about 9.5 mm. The thickness of the microwell array substrate may be uniform.

A variety of surface treatments and surface modification techniques may be used to alter the properties of microwell array surfaces. Examples can include, but are not limited to, oxygen plasma treatments to render hydrophobic material surfaces more hydrophilic, the use of wet or dry etching techniques to smooth (or roughen) glass and silicon surfaces, adsorption or grafting of polyethylene oxide or other polymer layers (such as pluronic), or bovine serum albumin to substrate surfaces to render them more hydrophilic and less prone to non-specific adsorption of biomolecules and cells, the use of silane reactions to graft chemically-reactive functional groups to otherwise inert silicon and glass surfaces, etc. Photodeprotection techniques can be used to selectively activate chemically-reactive functional groups at specific locations in the array structure, for example, the selective addition or activation of chemically-reactive functional groups such as primary amines or carboxyl groups on the inner walls of the microwells may be used to covalently couple oligonucleotide probes, peptides, proteins, or other biomolecules to the walls of the microwells. The choice of surface treatment or surface modification utilized can depend both or either on the type of surface property that is desired and on the type of material from which the microwell array is made.

The openings of microwells can be sealed, for example, during cell lysis steps to prevent cross hybridization of target nucleic acid between adjacent microwells. A microwell (or array of microwells) may be sealed or capped using, for example, a flexible membrane or sheet of solid material (i.e. a plate or platten) that clamps against the surface of the microwell array substrate, or a suitable bead, where the diameter of the bead is larger than the diameter of the microwell.

A seal formed using a flexible membrane or sheet of solid material can comprise, for example, inorganic nanopore membranes (e.g., aluminum oxides), dialysis membranes, glass slides, coverslips, elastomeric films (e.g. PDMS), or hydrophilic polymer films (e.g., a polymer film coated with a thin film of agarose that has been hydrated with lysis buffer).

Solid supports (e.g., beads) used for capping the microwells may comprise any of the solid supports (e.g., beads) of the disclosure. In some instances, the solid supports are cross-linked dextran beads (e.g., Sephadex). Cross-linked dextran can range from about 10 micrometers to about 80 micrometers. The cross-linked dextran beads used for capping can be from 20 micrometers to about 50 micrometers. In some embodiments, the beads may be at least about 10, 20, 30, 40, 50, 60, 70, 80 or 90% larger than the diameter of the microwells. The beads used for capping may be at most about 10, 20, 30, 40, 50, 60, 70, 80 or 90% larger than the diameter of the microwells.

The seal or cap may allow buffer to pass into and out of the microwell, while preventing macromolecules (e.g., nucleic acids) from migrating out of the well. A macromolecule of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides may be blocked from migrating into or out of the microwell by the seal or cap. A macromolecule of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides may be blocked from migrating into or out of the microwell by the seal or cap.

Solid supports (e.g., beads) may be distributed among a substrate. Solid supports (e.g., beads) can be distributed among wells of the substrate, removed from the wells of the substrate, or otherwise transported through a device comprising one or more microwell arrays by means of centrifugation or other non-magnetic means. A microwell of a substrate can be pre-loaded with a solid support. A microwell of a substrate can hold at least 1, 2, 3, 4, or 5, or more solid supports. A microwell of a substrate can hold at most 1, 2, 3, 4, or 5 or more solid supports. In some instances, a microwell of a substrate can hold one solid support.

Individual cells and beads may be compartmentalized using alternatives to microwells, for example, a single solid support and single cell could be confined within a single droplet in an emulsion (e.g. in a droplet digital microfluidic system).

Cells could potentially be confined within porous beads that themselves comprise the plurality of tethered stochastic barcodes. Individual cells and solid supports may be compartmentalized in any type of container, microcontainer, reaction chamber, reaction vessel, or the like.

Single cell combinatorial barcoding or may be performed without the use of microwells. Single cell, combinatorial barcoding assays may be performed without the use of any physical container. For example, combinatorial barcoding without a physical container can be performed by embedding cells and beads in close proximity to each other within a polymer layer or gel layer to create a diffusional barrier between different cell/bead pairs. In another example, combinatorial barcoding without a physical container can be performed in situ, in vivo, on an intact solid tissue, on an intact cell, and/or subcellularly.

Microwell arrays can be a consumable component of the assay system. Microwell arrays may be reusable. Microwell arrays can be configured for use as a stand-alone device for performing assays manually, or they may be configured to comprise a fixed or removable component of an instrument system that provides for full or partial automation of the assay procedure. In some embodiments of the disclosed methods, the bead-based libraries of stochastic barcodes can be deposited in the wells of the microwell array as part of the assay procedure. In some embodiments, the beads may be pre-loaded into the wells of the microwell array and provided to the user as part of, for example, a kit for performing stochastic barcoding and digital counting of nucleic acid targets.

In some embodiments, two mated microwell arrays are provided, one pre-loaded with beads which are held in place by a first magnet, and the other for use by the user in loading individual cells. Following distribution of cells into the second microwell array, the two arrays may be placed face-to-face and the first magnet removed while a second magnet is used to draw the beads from the first array down into the corresponding microwells of the second array, thereby ensuring that the beads rest above the cells in the second microwell array and thus minimizing diffusional loss of target molecules following cell lysis, while maximizing efficient attachment of target molecules to the stochastic barcodes on the bead.

Microwell arrays of the disclosure can be pre-loaded with solid supports (e.g., beads). Each well of a microwell array can comprise a single solid support. At least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the wells in a microwell array can be pre-loaded with a single solid support. At most 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the wells in a microwell array can be pre-loaded with a single solid support. The solid support can comprise stochastic barcodes and/or combinatorial barcodes of the disclosure. Cellular labels of stochastic barcodes on different solid supports can be different. Cellular labels of stochastic barcodes on the same solid support can be the same.

Three-Dimensional Substrates

A three-dimensional array can be any shape. A three-dimensional substrate can be made of any material used in a substrate of the disclosure. In some instances, a three-dimensional substrate comprises a DNA origami. DNA origami structures incorporate DNA as a building material to make nanoscale shapes. The DNA origami process can involve the folding of one or more long, “scaffold” DNA strands into a particular shape using a plurality of rationally designed “staple DNA strands. The sequences of the staple strands can be designed such that they hybridize to particular portions of the scaffold strands and, in doing so, force the scaffold strands into a particular shape. The DNA origami may include a scaffold strand and a plurality of rationally designed staple strands. The scaffold strand can have any sufficiently non-repetitive sequence.

The sequences of the staple strands can be selected such that the DNA origami has at least one shape to which stochastic labels can be attached. In some embodiments, the DNA origami can be of any shape that has at least one inner surface and at least one outer surface. An inner surface can be any surface area of the DNA origami that is sterically precluded from interacting with the surface of a sample, while an outer surface is any surface area of the DNA origami that is not sterically precluded from interacting with the surface of a sample. In some embodiments, the DNA origami has one or more openings (e.g., two openings), such that an inner surface of the DNA origami can be accessed by particles (e.g., solid supports). For example, in certain embodiments the DNA origami has one or more openings that allow particles smaller than 10 micrometers, 5 micrometers, 1 micrometer, 500 nm, 400 nm, 300 urn, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, 45 nm or 40 nm to contact an inner surface of the DNA origami.

The DNA origami can change shape (conformation) in response to one or more certain environmental stimuli. Thus an area of the DNA origami may be an inner surface when the DNA origami takes on some conformations, but may be an outer surface when the device takes on other conformations. In some embodiments, the DNA origami can respond to certain environmental stimuli by taking on a new conformation.

In some embodiments, the staple strands of the DNA origami can be selected such that the DNA origami is substantially barrel- or tube-shaped. The staples of the DNA origami can be selected such that the barrel shape is closed at both ends or is open at one or both ends, thereby permitting particles to enter the interior of the barrel and access its inner surface. In certain embodiments, the barrel shape of the DNA origami can be a hexagonal tube.

In some embodiments, the staple strands of the DNA origami can be selected such that the DNA origami has a first domain and a second domain, wherein the first end of the first domain is attached to the first end of the second domain by one or more single-stranded DNA hinges, and the second end of the first domain is attached to the second domain of the second domain by the one or more molecular latches. The plurality of staples can be selected such that the second end of the first domain becomes unattached to the second end of the second domain if all of the molecular latches are contacted by their respective external stimuli. Latches can be formed from two or more staple stands, including at least one staple strand having at least one stimulus-binding domain that is able to bind to an external stimulus, such as a nucleic acid, a lipid or a protein, and at least one other staple strand having at least one latch domain that binds to the stimulus binding domain. The binding of the stimulus-binding domain to the latch domain supports the stability of a first conformation of the DNA origami.

Synthesis of Combinatorial Barcodes on Solid Supports and Substrates

In some embodiments, a combinatorial barcode reagent can be synthesized on a solid support (e.g., bead). Pre-synthesized combinatorial barcode reagents (e.g., comprising the 5′amine that can link to the solid support) may be attached to solid supports (e.g., beads) through any of a variety of immobilization techniques involving functional group pairs on the solid support and the stochastic barcode. The combinatorial barcode reagent can comprise a functional group. The solid support (e.g., bead) can comprise a functional group. The combinatorial barcode reagent functional group and the solid support functional group can comprise, for example, biotin, streptavidin, primary amine(s), carboxyl(s), hydroxyl(s), aldehyde(s), ketone(s), and any combination thereof. A combinatorial barcode may be tethered to a solid support, for example, by coupling (e.g. using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) a 5′ amino group on the combinatorial barcode reagent to the carboxyl group of the functionalized solid support. Residual non-coupled combinatorial barcode reagents may be removed from the reaction mixture by performing multiple rinse steps. In some embodiments, the combinatorial barcode reagent and solid support are attached indirectly via linker molecules (e.g. short, functionalized hydrocarbon molecules or polyethylene oxide molecules) using similar attachment chemistries. The linkers may be cleavable linkers, e.g. acid-labile linkers or photo-cleavable linkers.

The combinatorial barcode reagents can be synthesized on solid supports (e.g., beads) using any of a number of solid-phase oligonucleotide synthesis techniques, such as phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, and phosphoramidite synthesis. Single nucleotides may be coupled in step-wise fashion to the growing, tethered combinatorial barcode reagent. In some embodiments, a short, pre-synthesized sequence (or block) of several oligonucleotides can be coupled to the growing, tethered combinatorial barcode reagent.

Combinatorial barcode reagents can be synthesized by interspersing step-wise or block coupling reactions with one or more rounds of split-pool synthesis, in which the total pool of synthesis beads is divided into a number of individual smaller pools which are then each subjected to a different coupling reaction, followed by recombination and mixing of the individual pools to randomize the growing combinatorial barcode reagent sequence across the total pool of beads. Split-pool synthesis is an example of a combinatorial synthesis process in which a maximum number of chemical compounds are synthesized using a minimum number of chemical coupling steps. The potential diversity of the compound library thus created is determined by the number of unique building blocks (e.g. nucleotides) available for each coupling step, and the number of coupling steps used to create the library. For example, a split-pool synthesis comprising 10 rounds of coupling using 4 different nucleotides at each step will yield 4¹⁰=1,048,576 unique nucleotide sequences. In some embodiments, split-pool synthesis may be performed using enzymatic methods such as polymerase extension or ligation reactions rather than chemical coupling. For example, in each round of a split-pool polymerase extension reaction, the 3′ ends of the stochastic barcodes tethered to beads in a given pool may be hybridized with the 5′ends of a set of semi-random primers, e.g. primers having a structure of 5′-(M)_(k)-(X)_(i)-(N)_(j)-3′, where (X)_(i) is a random sequence of nucleotides that is i nucleotides long (the set of primers comprising all possible combinations of (X)_(i)), (N)_(j) is a specific nucleotide (or series of j nucleotides), and (M)_(k) is a specific nucleotide (or series of k nucleotides), wherein a different deoxyribonucleotide triphosphate (dNTP) is added to each pool and incorporated into the tethered oligonucleotides by the polymerase.

The number of combinatorial barcode reagents conjugated to or synthesized on a solid support may comprise at least 100, 1000, 10000, or 1000000 or more combinatorial barcode reagents. The number of combinatorial barcode reagents conjugated to or synthesized on a solid support may comprise at most 100, 1000, 10000, or 1000000 or more combinatorial barcode reagents. The number of combinatorial barcode reagents conjugated to or synthesized on a solid support such as a bead may be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold more than the number of target nucleic acids in a cell. The number of combinatorial barcode reagents conjugated to or synthesized on a solid support such as a bead may be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold more than the number of target nucleic acids in a cell. At least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the stochastic barcode can be bound by a target nucleic acid. At most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the stochastic barcode can be bound by a target nucleic acid. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more different target nucleic acids can be captured by the combinatorial barcode reagents on the solid support. At most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more different target nucleic acids can be captured by the combinatorial barcode reagents on the solid support.

Methods of Barcoding a Plurality of Partitions

The disclosure provides for methods of barcoding a plurality of partitions with a set of combinatorial barcodes. In some embodiment, the plurality of partitions may be a plurality of microwells in a microwell array. In some embodiments, the number of mirowells in a microwell array can be at least 96, at least 384, at least 1,536, at least 5,000, at least 10,000, at least 25,000, at least 50,000, at least 75,000, at least 100,000, at least 500,000, at least 1,000,000, or at least 5,000,000 or more. In some embodiments, each microwell array may comprise an array barcode, distinguishable from the array barcode for another microwell array.

In some embodiments, the methods may comprise introducing a number of component barcodes in each of the plurality of partitions, wherein the component barcodes are selected from a set of component barcodes as disclosed herein. In some embodiments, the component barcodes in each of the plurality of partitions may be connected to each other to form a combinatorial barcode. The component barcodes may be connected to each other in the presence or absence of a target polynucleotide from a sample.

As described in FIG. 3 , combinatorial barcode reagents 305, 310, 315, and 320 can be dispensed into any particular microwell 325 (e.g., array of microwells 330). Dispensing can be performed, for example, by methods such as pipetting, pin spotting or non-contact printing. The combinatorial barcode reagents may also be added to wells randomly, for example, using delivery methods such as beads or other particles. For example, an inkjet print head can dispense combinatorial barcode reagents into each well. The barcoding reagents may be dispensed or created into individual partitions. Exemplary partitions can include, but are not limited to, a tube, a well in a microtiter plate, a flow cell, a hollow fiber, a microwell on slide, a hollow enclosed or open particle, an enclosed droplet, a particle such as a bead or other solid or liquid or gel particle or other physical partitions used to separate one sample from another. Physical isolation may also be achieved by the location separation of individual samples on a 2-dimensional surface or substrate in a 3-dimensional solid or scaffold.

Combinatorial barcode reagents can be designed in such a way so that each subunit code sequence from each subunit code section can be combined in a pre-programmed order using linkers. The final code in each microwell 325 can be unique. For any given combinatorial barcode reagent combination, the location of the microwell can be ascertained. In this way, combinatorial barcode reagents can be used to determine the spatial location of a sample in a microwell 325 on a microwell array 330.

The disclosure provides for compositions and methods for enabling the production of a massive repertoire of barcoding regent diversity using only a small set of component barcode reagents (e.g., combinatorial barcoding). The methods of combinatorial barcoding can be combined with the methods of stochastic barcoding. In some instances, hundreds of thousands to over millions of samples can be processed in parallel. For example, the number of samples that can be processed in parallel can be at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 thousands of samples. The number of samples that can be processed in parallel can be at most 100, 200, 300, 400, 500, 600, 700, 800, or 900 thousands of samples. The number of samples that can be processed in parallel can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more millions of samples. The number of samples that can be processed in parallel can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more millions of samples.

A method to simultaneously barcode tag the nucleic acids of many (thousands to millions) of individual samples is disclosed herein. The barcode tag may consist of one or more of a string of random or predetermined/known nucleic acid sequence (the barcode). The barcode can comprise a synthetic oligonucleotide and/or natural or unnatural DNA fragment. For example the barcode can be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length. The barcode can be, or be at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length.

In addition to different DNA sequences, the combinatorial barcodes may be comprised of different materials or come in different forms. For example, the component barcodes may be a combination of a bead and a gel particle, or of a first oligonucleotide in solution combined with a second oligonucleotide on a bead, or a first oligonucleotide immobilized on a substrate within a microwell and a second oligonucleotide in solution or present on a solid particle within the microwell. Barcodes may also be embedded within hydrogels or similar materials that can act as a sponge-like scaffold for the purpose of fixed position localization of biological samples and nucleic acids. For example, cells on a thin tissue section may be lysed and the released nucleic acid contents can be captured in position. The captured nucleic acids may incorporate position-specific barcodes or combinatorial barcodes, and be further replicated if desired and detected either in position or released and isolated for further characterization by methods such as next generation sequencing.

A sample can be dispensed into the microwells. In some embodiments, the sample can be a single cell. In some embodiments, the sample can be a tissue section. The sample can be dispensed prior to dispensing the combinatorial barcode reagents. The sample can be dispensed after dispensing the combinatorial barcode reagents. The sample can be dispensed with a dispensing (e.g., isolating) device. Exemplary dispensing/isolating devices can include, but are not limited to a flow cytometer, a needle array, and a microinjector. The microwell array can be attached to an isolating/dispensing device such that the sample can be isolated/dispensed directly into the wells of the microwell array.

Samples comprising the barcodes of the disclosure (i.e., samples that have undergone the barcoding method of the disclosure) can be used in downstream applications such as sequencing. After DNA sequencing, in addition to the sample nucleic acid sequences obtained, each sequence read can bear the sample barcode string that enables the assignment of that read to a particular sample within a pool of samples, for example, using computer processing.

A sample can be dispensed into the wells of a substrate in a non-random way. The location of each sample dispensed/isolated in the microwell array can be known. In this way, the distribution may not follow random and/or stochastic. A sample can be dispensed into the wells of a substrate in a non-Poisson way. A Poisson distribution or curve is a discrete probability distribution that expresses the probability of a number of events occurring in a fixed period of time if these events occur at a known average rate and are independent of one another. The Poisson distribution formula is as follows: f(k;λ)=(e^(−λ)λ^(k)/k!) where k is the number of occurrences of an event and λ is a positive real number of the expected number of occurrences during the given interval. Non-Poisson distribution can be a distribution that does not follow the Poisson equation.

The microwells comprising the nucleic acids, the single cell, nucleic acids from a single cell, the combinatorial barcode reagents, and reagents necessary for primer extension and amplification (e.g., polymerase dNTPs, buffer), or any combination thereof, can be sealed. Sealing of the microwells may be useful for preventing cross hybridization of target nucleic acid between adjacent microwells. A microwell can be sealed with tape and/or any adhesive. The sealant may be clear. The sealant may be opaque. The sealant may allow light to pass through thereby enabling visual detection of the contents of the sealed microwell (e.g., UV-Vis, fluorescence). Nucleic acids (e.g., from the sample) in the partitions (e.g., microwells of an array) can associate (e.g., hybridize) with the combinatorial barcode reagents dispensed in the microwell.

The combinatorial barcode reagents and target polynucleotides inside a well can be extended and/or amplified to generate a transcript and/or amplicon that comprises the sequence of the combinatorial barcode reagents. Amplification can be performed by methods, including, but not limited to PCR, primer extension, reverse transcription, isothermal amplification, linear amplification, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR, end-point PCR, and suppression PCR, or any combination thereof. In some instances, amplification and/or extension is performed with isothermal amplification. In some instances, amplification and/or extension is performed with multiple strand displacement.

The contents of the microwells can be pooled after extension and/or amplification. Pooling the contents can reduce errors in the sample preparation for downstream processes. Microwell samples can be pooled into one container (e.g., tube). Microwell samples can be pooled into more than one container (e.g., tube for sample/plate indexing).

Pooled microwell samples can be manipulated with molecular biology. For example, the pooled samples can be subjected to one or more rounds of amplification. The pooled samples can be subjected to a purification step (e.g., with Ampure beads, size selection, gel/column filtration, removal of rRNA or any other RNA impurity, enzymatic degradation of impurities, pulse gel electrophoresis, and sedimentation through a sucrose gradient or a cesium chloride gradient, and size exclusion chromatography (gel-permeation chromatography).

The pooled microwell samples can be prepared for sequencing. Sequencing preparation can comprise the addition of sequencing (e.g., flowcell adaptors) either through adaptor ligation (e.g., TA ligation) or primer extension (e.g., wherein primers comprise the sequences of the flowcell adaptors). Methods for ligating adaptors to fragments of nucleic acid are well known. Adaptors may be double-stranded, single-stranded or partially single-stranded. In some aspects, adaptors are formed from two oligonucleotides that have a region of complementarity, for example, about 10 to 30, or about 15 to 40 bases of perfect complementarity; so that when the two oligonucleotides are hybridized together they form a double stranded region. Optionally, either or both of the oligonucleotides may have a region that is not complementary to the other oligonucleotide and forms a single stranded overhang at one or both ends of the adaptor. Single-stranded overhangs may be about 1 to about 8 bases, or about 2 to about 4. The overhang may be complementary to the overhang created by cleavage with a restriction enzyme to facilitate “sticky-end” ligation. Adaptors may include other features, such as primer binding sites and restriction sites. In some aspects the restriction site may be for a Type IIS restriction enzyme or another enzyme that cuts outside of its recognition sequence, such as EcoP151. The pooled samples can be sequenced (e.g., by methods described herein).

Sequencing the combinatorially barcoded nucleic acids can comprise sequencing at least a portion of the combinatorial barcode (e.g., the sequences of the combinatorial barcode reagents used to make the combinatorial barcode). Sequencing can comprise sequencing at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the combinatorial barcode of the target polynucleotide. Sequencing can comprise sequencing at most 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the combinatorial barcode of the target polynucleotide. Sequencing can comprise sequencing at least a portion of the target polynucleotide. Sequencing can comprise sequencing at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the target polynucleotide. Sequencing can comprise sequencing at most 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the target polynucleotide. Sequencing can comprise sequencing at least a portion of the combinatorial barcode and at least a portion of the target polynucleotide. Sequencing can comprise sequencing the junction between the combinatorial barcode and target polynucleotide. Sequencing can comprise paired-end sequencing. Paired-end sequencing can allow the determination of two or more reads of sequence from two places on a single polynucleotide duplex.

After DNA sequencing, in addition to the sample nucleic acid sequences obtained, each sequence read can bear the sample barcode string and/or a target nucleic acid sequence that enables the assignment of that read to a particular sample within a pool of samples, for example, using computer processing. For example, reads that comprise a combinatorial barcode sequence from a first microwell can be binned together while reads that comprise a combinatorial barcode sequence from a second microwell can be binned together. The reads for each microwell (e.g., bin) can be analyzed and/or processed. For example, when the starting material was fragmented chromosomal DNA, the reads can be processed to form contigs and/or haplotype phasing can be performed as described in PCT Application No. PCT/US2016/22712, the content of which is incorporated by reference in its entirety.

For example, contigs (continuous sequences) can be formed from the short-range characterization data of the reads. Reads can be binned. In every bin, the short-range probe signatures of the reads in that bin can be retrieved and recored. Using conventional contiging analysis on subsets of binned reads, the short-range signatures can be used to determine read orders and distances for the contiged reads in the bin. The contigs of neighboring bins can be oriented and connected to form larger contigs.

In some embodiments, reads are binned according to short-range probings. Following initial comparisons of short-range probe signatures, clusters with high confidence of reads are formed. The read order and distances are then determined for the reads in the cluster. The long-range probe information for every read cluster is retrieved and recorded, and a composite long-range score for the cluster is formed. This composite can be formed for each entry by taking the maximum (or other arithmetic combination) of the (read) comparison scores over all the reads in the cluster. The result is a binning of the cluster relative to the genome, and this global positioning information can be used to determine neighboring clusters. The contigs of neighboring binned clusters can be then oriented and connected to form larger contigs.

Barcoding a Plurality of DNA Targets

Some embodiments disclosed herein provide methods of barcoding a plurality of DNA targets in a plurality of partitions. The plurality of partitions can be barcoded using the combinatorial barcodes as disclosed herein. For example, each partition may be barcoded with a unique combination of component barcodes selected from a set of component barcodes. In some embodiments, each combinatorial barcode in each of the plurality of partitions comprises a target-specific region that binds to one or more DNA targets in the partition. A plurality of DNA targets, such as genomic DNA fragments, may be introduced to the plurality of partitions. In some embodiments, each partition comprises no more than 1 DNA target. In some embodiments, each partition comprises no more than 1 DNA target from the same chromosome. In some embodiments, each partition may comprise on average no more than 1, no more than 2, no more than 3, no more than 5, no more than 10, no more than 100 DNA targets. In some embodiments, the DNA targets are introduced to the partitions after the partitions have been introduced with the component barcodes. In some embodiments, the DNA targets are introduced to the partitions before the partitions have been introduced with the component barcodes. In some embodiments, one or more of the component barcodes in each partition hybridize to the one or more DNA targets in the partition. In some embodiments, the component barcode that hybridized to the DNA target may be used as a primer for an extension reaction to produce an extension product comprising the DNA target and the combinatorial barcode. In some embodiments, the extension product may be amplified and sequenced.

FIG. 4 depicts an exemplary embodiment of the combinatorial barcoding method of the disclosure to generate contigs. A nucleic acid (e.g., chromosome) can be fragmented. Fragmentation can occur by any method, such as for example, sonication, shearing, and enzymatic fragmentation. The fragments can be, or be at least, 10, 50, 100, 150, 200, 250, 300, 350, or 400 kilobases or more in length. The fragments can be at most 10, 50, 100, 150, 200, 250, 300, 350, or 400 kilobases or more in length. The fragmenting can produce at least 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, or 1×10⁸ or more fragments. The fragmenting can produce at most 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, or 1×10⁸ or more fragments. The fragments isolated in the well can be about 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-15, or 10-20 fragments per well. The fragments isolated in the wells can be overlapping. The fragments isolated in the wells can be non-overlapping. Nucleic acid fragments 405 can be isolated into wells 410 of a microwell array 415. Some or all of the wells of the microwell array can comprise one or more combinatorial barcode reagents 420 and 425 (e.g., a known grouping of combinatorial barcode reagents, thereby generating a unique combinatorial barcode). The microwells can be sealed. The contents of the microwells can be extended/amplified (e.g., by primer extension and/or multiple strand displacement) thereby generating a transcript comprising the combinatorial barcode 430. The transcript/amplicon comprising the combinatorial barcode 430 can be sequenced. Sequencing analysis can comprise binning reads based on their combinatorial barcode sequence, which correspond to the same microwell. The reads can be generated into contigs. The contigs can be used for haplotype phasing (e.g., of parental SNPs).

Single Cell Sequencing

Some embodiments disclosed herein provide methods of single cell sequencing. The plurality of partitions may be barcoded using the combinatorial barcodes as disclosed herein. For example, each partition may be barcoded with a unique combination of component barcodes selected from a set of component barcodes. In some embodiments, each combinatorial barcode in each of the plurality of partitions comprises a target-specific region that binds to one or more target polynucleotides of the single cell in the partition. In some embodiments, the target polynucleotides comprise DNA. In some embodiments, the target polynucleotides comprise RNA.

A plurality of single cells can be introduced into a plurality of partitions, such as microwells of a microwell array. In some embodiments, the cells may be enriched for desirable characteristics using a flow cytometer. In some embodiments, introducing the plurality of single cells into the microwells of the microwell array can comprise flow cytometrically depositing the plurality of cells into the microwells of the microwell array. Flow cytometrically depositing the plurality of single cells into the microwells of the microwell array can comprise using a flow cytometer to deposit a single cell at a time into the microwells of the microwell array. In some embodiments, the plurality of partitions is comprised of greater than 1536 microwells. In some embodiments, the plurality of partitions is a microwell array format of 5 micrometers or more. In some embodiments, the plurality of partitions is defined dimensionally, such as by spatial positioning.

FIG. 5 illustrates an exemplary method for single cell sequencing. The sample to be barcoded can comprise single cells and/or nucleic acid from single cells. Single cells 505 can be isolated into wells 510 of a microwell array 515. In some embodiments, at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the wells can comprise a single cell. In some embodiments, at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the wells can comprise a single cell. Cells can be distributed to a well in a non-Poisson manner. Cells can be distributed to wells in a non-random manner. Cells can be distributed to wells in a Poisson manner. Cells can be distributed to wells in a random manner.

The cells can be lysed, thereby releasing nucleic acid contents of the cells. The wells can be sealed prior to lysis. The nucleic acid contents of the cells can associate with one or more combinatorial barcode reagents in the microwell 520/525 (e.g., a known grouping of combinatorial barcode reagents, thereby generating a unique combinatorial barcode). The contents of the microwells can be extended/amplified (e.g., by primer extension and/or multiple strand displacement) thereby generating a transcript comprising the combinatorial barcode 530. The transcript/amplicon comprising the combinatorial barcode 530 can be sequenced. Sequencing analysis can comprise binning reads based on their combinatorial barcode sequence, which correspond to the same microwell. The reads can be analyzed for gene expression analysis of the single cell. The reads can be used to analyze the cell (e.g., determine the cell type, diagnose the cell as part of a disorder or disease, genotype the cell, determine or predict the response of the cell to a therapy or regimen, and/or measure changes in the cell's expression profile over time).

In some instances, the methods of the disclosure can comprise stochastic barcoding and combinatorial barcoding. For example, and shown in FIG. 6 , cells 605 can be distributed to wells 610 a microwell array 615 in a non-Poisson manner, such that a well contains a single cell. The cells can be lysed. The nucleic acids 620 from the cells can be contacted to a plurality of stochastic barcodes 625 comprising an oligo dT and cellular label, and molecular label of the disclosure can be contacted to a target (e.g., mRNA). The stochastic barcode can be reverse transcribed, thereby generating a stochastically barcoded cDNA 630. The stochastically barcoded cDNA can be contacted with one or more combinatorial barcode reagents 635/640 that can perform second strand synthesis. The one or more combinatorial barcode reagents 635/640 for second strand synthesis can comprise a universal sequence (e.g., a sequencing primer binding site, i.e., Illumina read 1). Second strand synthesis can result in a stochastically barcoded and combinatorially barcoded nucleic acid molecule 645. The stochastically barcoded and combinatorially barcoded nucleic acid molecule 645 can be subjected to downstream library preparation methods (e.g., pooling, sequencing adaptor addition) and sequenced. The stochastic barcode can be used to count the number of target nucleic acids 620 in the sample 605. The combinatorial barcode can be used to identify the well 610 of the microwell array 615 that the cell came from.

In some instances, the methods of the disclosure can comprise bipartite combinatorial barcoding. For example, and shown in FIG. 7 , cells 705 can be distributed to wells 710 a microwell array 715 in a non-Poisson manner, such that a well contains a single cell. The cells can be lysed. The nucleic acids 720 from the cells can be contacted to one or more 3′ combinatorial barcode reagents 725 can be contacted to a target (e.g., mRNA). The one or more 3′ combinatorial barcode reagents 725 can be reverse transcribed, thereby generating a 3′combinatorial barcoded cDNA 730. The 3′combinatorial barcoded cDNA 730 can be contacted with one or more 5′ combinatorial barcode reagents 740 that can perform second strand synthesis. The one or more 5′ combinatorial barcode reagents 740 for second strand synthesis can comprise a universal sequence (e.g., a sequencing primer binding site, i.e., Illumina read 1). Second strand synthesis can result in a bipartite combinatorially barcoded nucleic acid molecule 745. The bipartite combinatorially barcoded nucleic acid molecule 745 can be subjected to downstream library preparation methods (e.g., pooling, sequencing adaptor addition) and sequenced. The bipartite combinatorial barcode can be used to identify the well 710 of the microwell array 715 that the cell came from.

The methods disclosed herein can be used to compare cells and/or nucleic acid transcripts at varying time points. The methods of the disclosure can be used to diagnose a subject, or compare the cells/nucleic acids of a diseased subject with a healthy subject. For example, each well of a microwell array can comprise a cell from a different subject.

The methods disclosed herein can be used, for example, in pharmaceutical testing or clinical trial testing. For example, the microwells of the substrate comprising the combinatorial barcode reagents can comprise a modulating reagent. A modulating reagent can comprise a drug, a therapy, a protein, an RNA, a liposome, a lipid, or any therapeutic molecule (e.g., clinical trial molecule, molecule approved by the FDA, molecule not approved by the FDA). A modulating agent can be introduced into a well prior to isolation of the sample in the well. A modulating agent can be introduced into a well after isolation of the sample in the well (e.g., the modulating agent can be contacted to samples in the well). In one example, each well of a microwell array can comprise a cell from different subjects. The cells can be treated with a modulating agent. The sequencing results can be used to determine how the modulating agent affected gene expression of the sample (e.g., single cell). The sequencing results can be used to determine which subject from a population of subjects may be affected by the modulating agent and/or diagnose a subject in the population.

Methods of Stochastic Barcoding

The disclosure provides for methods for combinatorial barcoding and/or stochastic barcoding of a sample. The methods of combinatorial barcoding and stochastic barcoding can be combined. The methods can comprise placing the stochastic barcodes in close proximity with the sample, lysing the sample, associating distinct targets with the stochastic barcodes, amplifying the targets and/or digitally counting the targets. The method can further comprise analyzing and/or visualizing the information obtained from the spatial labels on the stochastic barcodes. A sample (e.g., section of a sample, thin slice, cell) can be contacted with a solid support comprising a stochastic barcode. Targets in the sample can be associated with the stochastic barcodes. The solid supports can be collected. cDNA synthesis can be performed on the solid support. cDNA synthesis can be performed off the solid support. cDNA synthesis can incorporate the label information from the labels in the stochastic barcode into the new cDNA target molecule being synthesized, thereby generating a target-barcode molecule. The target-barcode molecules can be amplified using PCR. The sequence of the targets and the labels of the stochastic barcode on the target-barcode molecule can be determined by sequencing methods.

Contacting a Sample and a Stochastic Barcode

The disclosure provides for methods of distributing a sample (e.g., a cell) to a plurality of wells of a substrate, wherein the wells comprise combinatorial barcode reagents, and wherein the distributing occurs in a non-Poisson manner. The distribution/isolation of cells into the wells can be non-random (e.g., cells are specifically sorted into specific locations in the array). In this way, the distribution of cells can be considered non-Poisson.

A sample comprising, for example, a cell, organ, or tissue thin section, can be contacted to stochastic barcodes. The solid supports can be free floating. The solid supports can be embedded in a semi-solid or solid array. The stochastic barcodes may not be associated with solid supports. The stochastic barcodes may be individual nucleotides. The stochastic barcodes may be associated with a substrate. When stochastic barcodes are in close proximity to targets, the targets can hybridize to the stochastic barcode. The stochastic barcodes can be contacted at a non-depleatable ratio such that each distinct target can associate with a distinct stochastic barcode of the disclosure. To ensure efficient association between the target and the stochastic barcode, the targets can be crosslinked to the stochastic barcode.

The probability that two distinct targets of a sample can contact the same unique stochastic barcode can be, or be at least, 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹ or more. The probability that two distinct targets of a sample can contact the same unique stochastic barcode can be at most 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹ or more. The probability that two targets of the same gene from the same cell can contact the same stochastic barcode can be, or be at least, 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹ or more. The probability that two targets of the same gene from the same cell can contact the same stochastic barcode can be at most 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹ or more.

In some instances, cells from a population of cells can be separated (e.g., isolated) into wells of a substrate of the disclosure. The population of cells can be diluted prior to separating. The population of cells can be diluted such that at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of wells of the substrate receive a single cell. The population of cells can be diluted such that at most 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of wells of the substrate receive a single cell. The population of cells can be diluted such that the number of cells in the diluted population is, or is at least, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the number of wells on the substrate. The population of cells can be diluted such that the number of cells in the diluted population is, or is at least, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the number of wells on the substrate. In some instances, the population of cells is diluted such that the number of cell is about 10% of the number of wells in the substrate.

Distribution of single cells into wells of the substrate can follow a Poisson distribution. For example, there can be at least a 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% or more probability that a well of the substrate has more than one cell. There can be at least a 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% or more probability that a well of the substrate has more than one cell. Distribution of single cells into wells of the substrate can be random. Distribution of single cells into wells of the substrate can be non-random. The cells can be separated such that a well of the substrate receives only one cell.

Cell Lysis

Following the distribution of cells and stochastic barcodes, the cells can be lysed to liberate the target molecules. Cell lysis may be accomplished by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis. Cells may be lysed by addition of a cell lysis buffer comprising a detergent (e.g. SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g. methanol or acetone), or digestive enzymes (e.g. proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of a target and a stochastic barcode, the rate of the diffusion of the target molecules can be altered by for example, reducing the temperature and/or increasing the viscosity of the lysate.

Attachment of Stochastic Barcodes to Target Nucleic Acid Molecules

Following lysis of the cells and release of nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with the stochastic barcodes of the co-localized solid support. Association can, for example, comprise hybridization of a stochastic barcode's target recognition region to a complementary portion of the target nucleic acid molecule (e.g., oligo dT of the stochastic barcode can interact with a poly-A tail of a target). The assay conditions used for hybridization (e.g. buffer pH, ionic strength, temperature, etc.) can be chosen to promote formation of specific, stable hybrids.

Attachment can further comprise ligation of a stochastic barcode's target recognition region and a portion of the target nucleic acid molecule. For example, the target binding region may comprise a nucleic acid sequence that can be capable of specific hybridization to a restriction site overhang (e.g. an EcoRI sticky-end overhang). The assay procedure can further comprise treating the target nucleic acids with a restriction enzyme (e.g. EcoRI) to create a restriction site overhang. The stochastic barcode may then be ligated to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) may be used to join the two fragments.

The labeled targets from a plurality of cells (or a plurality of samples) (e.g., target-barcode molecules) can be subsequently pooled, for example by retrieving the stochastic barcodes and/or the beads to which the target-barcode molecules are attached. The retrieval of solid support-based collections of attached target-barcode molecules may be implemented by use of magnetic beads and an externally-applied magnetic field. Once the target-barcode molecules have been pooled, all further processing may proceed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and/or nucleic acid extension reactions. Further processing reactions may be performed within the microwells, that is, without first pooling the labeled target nucleic acid molecules from a plurality of cells.

Reverse Transcription

The disclosure provides for a method to create a stochastic target-barcode conjugate using reverse transcription. The stochastic target-barcode conjugate can comprise the stochastic barcode and a complementary sequence of all or a portion of the target nucleic acid (i.e. a stochastically barcoded cDNA molecule). Reverse transcription of the associated RNA molecule may occur by the addition of a reverse transcription primer along with the reverse transcriptase. The reverse transcription primer can be an oligo-dT primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Oligo-dT primers can be 12-18 nucleotides in length and bind to the endogenous poly-A tail at the 3′ end of mammalian mRNA. Random hexanucleotide primers may bind to mRNA at a variety of complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

Amplification

One or more nucleic acid amplification reactions may be performed to create multiple copies of the labeled target nucleic acid molecules. Amplification may be performed in a multiplexed manner, wherein multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction may be used to add sequencing adaptors to the nucleic acid molecules. The amplification reactions may comprise amplifying at least a portion of a sample label, if present. The amplification reactions may comprise amplifying at least a portion of the cellular and/or molecular label. The amplification reactions may comprise amplifying at least a portion of a sample tag, a cellular label, a spatial label, a molecular label, a target nucleic acid, or a combination thereof. The amplification reactions may comprise amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids. The method may further comprise conducting one or more cDNA synthesis reactions to produce one or more cDNA copies of target-barcode molecules comprising a sample label, a cellular label, a spatial label, and/or a molecular label.

In some embodiments, amplification may be performed using a polymerase chain reaction (PCR). As used herein, PCR may refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. As used herein, PCR may encompass derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR.

Amplification of the labeled nucleic acids can comprise non-PCR based methods. Examples of non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), whole transcriptome amplification (WTA), whole genome amplification (WGA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, a ligase chain reaction (LCR), and a Qβ replicase (Qβ) method, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using a restriction endonuclease, an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to the extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5′ exonuclease activity, rolling circle amplification, and ramification extension amplification (RAM). In some instances, the amplification may not produce circularized transcripts.

Suppression PCR can be used for amplification methods of the disclosure. Suppression PCR can refer to the selective exclusion of molecules less than a certain size flanked by terminal inverted repeats, due to their inefficient amplification when the primer(s) used for amplification correspond(s) to the entire repeat or a fraction of the repeat. The reason for this can lie in the equilibrium between productive PCR primer annealing and nonproductive self-annealing of the fragment's complementary ends. At a fixed size of a flanking terminal inverted repeat, the shorter the insert, the stronger the suppression effect and vice versa. Likewise, at a fixed insert size, the longer the terminal inverted repeat, the stronger the suppression effect.

Suppression PCR can use adapters that are ligated to the end of a DNA fragment prior to PCR amplification. Upon melting and annealing, single-stranded DNA fragments having self-complementary adapters at the 5′- and 3′-ends of the strand can form suppressive “tennis racquet” shaped structures that suppress amplification of the fragments during PCR.

In some instances, the methods disclosed herein further comprise conducting a polymerase chain reaction on the labeled nucleic acid (e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to produce a stochastically labeled-amplicon. The labeled-amplicon may be double-stranded molecule. The double-stranded molecule may comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or a RNA molecule hybridized to a DNA molecule. One or both of the strands of the double-stranded molecule may comprise a sample label, a spatial label, a cellular label, and/or a molecular label. The stochastically labeled-amplicon can be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the disclosure may comprise synthetic or altered nucleic acids.

Amplification may comprise use of one or more non-natural nucleotides. Non-natural nucleotides may comprise photolabile or triggerable nucleotides. Examples of non-natural nucleotides can include, but are not limited to, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Non-natural nucleotides may be added to one or more cycles of an amplification reaction. The addition of the non-natural nucleotides may be used to identify products as specific cycles or time points in the amplification reaction.

Conducting the one or more amplification reactions may comprise the use of one or more primers. The one or more primers may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may comprise less than 12-15 nucleotides. The one or more primers may anneal to at least a portion of the plurality of stochastically labeled targets. The one or more primers may anneal to the 3′ end or 5′ end of the plurality of stochastically labeled targets. The one or more primers may anneal to an internal region of the plurality of stochastically labeled targets. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3′ ends the plurality of stochastically labeled targets. The one or more primers may comprise a fixed panel of primers. The one or more primers may comprise at least one or more custom primers. The one or more primers may comprise at least one or more control primers. The one or more primers may comprise at least one or more gene-specific primers.

The one or more primers may comprise any universal primer of the disclosure. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample label, a second sample label, a spatial label, a cellular label, a molecular label, a target, or any combination thereof. The one or more primers may comprise a universal primer and a custom primer. The custom primer may be designed to amplify one or more targets. The targets may comprise a subset of the total nucleic acids in one or more samples. The targets may comprise a subset of the total stochastically labeled targets in one or more samples. The one or more primers may comprise at least 96 or more custom primers. The one or more primers may comprise at least 960 or more custom primers. The one or more primers may comprise at least 9600 or more custom primers. The one or more custom primers may anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids may correspond to one or more genes.

Any amplification scheme can be used in the methods of the present disclosure. For example, in one scheme, the first round PCR can amplify molecules (e.g., attached to the bead) using a gene specific primer and a primer against the universal Illumina sequencing primer 1 sequence. The second round of PCR can amplify the first PCR products using a nested gene specific primer flanked by Illumina sequencing primer 2 sequence, and a primer against the universal Illumina sequencing primer 1 sequence. The third round of PCR adds P5 and P7 and sample index to turn PCR products into an Illumina sequencing library. Sequencing using 150 bp×2 sequencing can reveal the cell label and molecular index on read 1, the gene on read 2, and the sample index on index 1 read.

Amplification can be performed in one or more rounds. In some instances there are multiple rounds of amplification. Amplification can comprise two or more rounds of amplification. The first amplification can be an extension off X′ to generate the gene specific region. The second amplification can occur when a sample nucleic hybridizes to the newly generated strand.

In some embodiments hybridization does not need to occur at the end of a nucleic acid molecule. In some embodiments a target nucleic acid within an intact strand of a longer nucleic acid is hybridized and amplified. For example a target within a longer section of genomic DNA or mRNA. A target can be more than 50 nt, more than 100 nt, or more that 1000 nt from an end of a polynucleotide.

Sequencing

Determining the number of different stochastically labeled nucleic acids may comprise determining the sequence of the labeled target, the spatial label, the molecular label, the sample label, and the cellular label or any product thereof (e.g. labeled-amplicons, labeled-cDNA molecules). An amplified target can be subjected to sequencing. Determining the sequence of the stochastically labeled nucleic acid or any product thereof may comprise conducting a sequencing reaction to determine the sequence of at least a portion of a sample label, a spatial label, a cellular label, a molecular label, and/or at least a portion of the stochastically labeled target, a complement thereof, a reverse complement thereof, or any combination thereof.

Determination of the sequence of a nucleic acid (e.g. amplified nucleic acid, labeled nucleic acid, cDNA copy of a labeled nucleic acid, etc.) can be performed using variety of sequencing methods including, but not limited to, sequencing by synthesis (SBS) sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads, wobble sequencing, multiplex sequencing, polymerized colony (POLONY) sequencing; nanogrid rolling circle sequencing (ROLONY), allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout), and the like.

In some instances, determining the sequence of the labeled nucleic acid or any product thereof comprises paired-end sequencing, nanopore sequencing, high-throughput sequencing, shotgun sequencing, dye-terminator sequencing, multiple-primer DNA sequencing, primer walking, Sanger dideoxy sequencing, Maxim-Gilbert sequencing, pyrosequencing, true single molecule sequencing, or any combination thereof. Alternatively, the sequence of the labeled nucleic acid or any product thereof may be determined by electron microscopy or a chemical-sensitive field effect transistor (chemFET) array.

High-throughput sequencing methods, such as cyclic array sequencing using platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, or the Polonator platform, may also be utilized. Sequencing may comprise MiSeq sequencing. Sequencing may comprise HiSeq sequencing.

The stochastically labeled targets can comprise nucleic acids representing from about 0.01% of the genes of an organism's genome to about 100% of the genes of an organism's genome. For example, about 0.01% of the genes of an organism's genome to about 100% of the genes of an organism's genome can be sequenced using a target complementary region comprising a plurality of multimers by capturing the genes containing a complementary sequence from the sample. In some embodiments, the labeled nucleic acids comprise nucleic acids representing from about 0.01% of the transcripts of an organism's transcriptome to about 100% of the transcripts of an organism's transcriptome. For example, about 0.501% of the transcripts of an organism's transcriptome to about 100% of the transcripts of an organism's transcriptome can be sequenced using a target complementary region comprising a poly-T tail by capturing the mRNAs from the sample.

Sequencing can comprise sequencing at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides or base pairs of the labeled nucleic acid and/or stochastic barcode. Sequencing may comprise sequencing at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides or base pairs of the labeled nucleic acid and/or stochastic barcode. Sequencing can comprise sequencing at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more nucleotides or base pairs of the labeled nucleic acid and/or stochastic barcode. Sequencing can comprise sequencing at most about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more nucleotides or base pairs of the labeled nucleic acid and/or stochastic barcode. Sequencing can comprise sequencing at least about 1,500; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more nucleotides or base pairs of the labeled nucleic acid and/or stochastic barcode. Sequencing can comprise sequencing at most about 1,500; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more nucleotides or base pairs of the labeled nucleic acid and/or stochastic barcode.

Sequencing can comprise at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more sequencing reads per run. Sequencing may comprise at most about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more sequencing reads per run. In some instances, sequencing comprises sequencing at least about 1,500; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more sequencing reads per run. In some instances, sequencing comprises sequencing at most about 1,500; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more sequencing reads per run. Sequencing can comprise sequencing at least 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 or more millions of sequencing reads per run. Sequencing can comprise sequencing at most 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 or more millions of sequencing reads per run. Sequencing can comprise sequencing at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 2000, 3000, 4000, or 5000 or more millions of sequencing reads in total. Sequencing can comprise sequencing at most 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 2000, 3000, 4000, or 5000 or more millions of sequencing reads in total. Sequencing may comprise less than or equal to about 1,600,000,000 sequencing reads per run. Sequencing may comprise less than or equal to about 200,000,000 reads per run.

Methods of Spatial Barcoding

Some embodiments disclosed herein provide methods of spatial barcoding of nucleic acids in a target from a sample. In some embodiments, a plurality of oligonucleotides is immobilized to a substrate, such as a slide. In some embodiments, the substrate may be coated with a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the oligonucleotides comprise a target-specific region, such as an oligo(dT) sequence, a gene-specific sequence, a random multimer, etc.

A sample comprising, for example, a slice, a cell monolayer, fixed cells, a tissue section, can be contacted to the oligonucleotides on the substrate. The cells may comprise one or more cell types. For example, the cells can be brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, circulatory cells, or any combination thereof. The cells can be contacted, for example, by gravity flow wherein the cells can settle and create a monolayer. The sample can be a tissue thin section. The thin section can be placed on the substrate. The sample can be one-dimensional (e.g., form a planar surface). The sample (e.g., cells) can be spread across the substrate, for example, by growing/culturing the cells on the substrate.

Cell Lysis

Following the distribution of cells and stochastic barcodes, the cells can be lysed to liberate the target molecules. Cell lysis can be accomplished by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis. Cells can be lysed by addition of a cell lysis buffer comprising a detergent (e.g. SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g. methanol or acetone), or digestive enzymes (e.g. proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of a target and a stochastic barcode, the rate of the diffusion of the target molecules can be altered by for example, reducing the temperature and/or increasing the viscosity of the lysate.

In some embodiments, the sample can be lysed using a filter paper. The filter paper can be soaked with a lysis buffer on top of the filter paper. The filter paper can be applied to the sample with pressure which can facilitate lysis of the sample and hybridization of the targets of the sample to the substrate.

In some embodiments, lysis can be performed by mechanical lysis, heat lysis, optical lysis, and/or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by the addition of a lysis buffer to the substrate. A lysis buffer can comprise Tris HCl. A lysis buffer can comprise at least about 0.01, 0.05, 0.1, 0.5, or 1M or more Tris HCl. A lysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1M or more Tris HCL. A lysis buffer can comprise about 0.1 M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). The concentration of salt in the lysis buffer can be at least about 0.1, 0.5, or 1M or more. The concentration of salt in the lysis buffer can be at most about 0.1, 0.5, or 1M or more. In some embodiments, the concentration of salt in the lysis buffer is about 0.5M. The lysis buffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, triton X, tween, NP-40). The concentration of the detergent in the lysis buffer can be at least about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or 7% or more. The concentration of the detergent in the lysis buffer can be at most about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or 7% or more. In some embodiments, the concentration of the detergent in the lysis buffer is about 1% Li dodecyl sulfate. The time used in the method for lysis can be dependent on the amount of detergent used. In some embodiments, the more detergent used, the less time needed for lysis. The lysis buffer can comprise a chelating agent (e.g., EDTA, EGTA). The concentration of a chelating agent in the lysis buffer can be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of a chelating agent in the lysis buffer can be at most about 1, 5, 10, 15, 20, 25, or 30 mM or more. In some embodiments, the concentration of chelating agent in the lysis buffer is about 10 mM. The lysis buffer can comprise a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer can be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer can be at most about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of reducing reagent in the lysis buffer is about 5 mM. In some embodiments, a lysis buffer can comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT. [0087] Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or 30 C. Lysis can be performed for about 1, 5, 10, 15, or 20 or more minutes. A lysed cell can comprise at least about 100000, 200000, 300000, 400000, 500000, 600000, or 700000 or more target nucleic acid molecules. A lysed cell can comprise at most about 100000, 200000, 300000, 400000, 500000, 600000, or 700000 or more target nucleic acid molecules.

In some embodiments, the oligonucleotides can hybridize to the nucleic acids released from the cells. The nucleic acids can comprise DNAs, such as genomic DNAs, or RNAs, such as messenger RNAs (mRNAs), microRNAs, small interfering RNAs (siRNAs), RNA degradation products, RNAs each comprising a poly(A) tail, or any combination thereof. The nucleic acids hybridized to the oligonucleotides can be used as a template for an extension reaction, such as reverse transcription, DNA polymerization, etc.

The nucleic acid molecules released from the lysed cells can associate with the plurality of probes on the substrate (e.g., hybridize with the probes on the substrate). When the probes comprise oligo dT, mRNA molecules can hybridize to the probes and be reverse transcribed. The oligodT portion of the oligonucleotide can act as a primer for first strand synthesis of the cDNA molecule. Reverse transcription of the labeled-RNA molecule can occur by the addition of a reverse transcription primer. In some instances, the reverse transcription primer is an oligodT primer, random hexanucleotide primer, or a target-specific oligonucleotide primer. Generally, oligodT primers are 12-18 nucleotides in length and bind to the endogenous poly(A)+ tail at the 3′ end of mammalian mRNA. Random hexanucleotide primers may bind to mRNA at a variety of complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

Reverse transcription can occur repeatedly to produce multiple labeled-cDNA molecules. The methods disclosed herein may comprise conducting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The method may comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

In some embodiments, nucleic acids can be removed from the substrate using chemical cleavage. For example, a chemical group or a modified base present in a nucleic acid can be used to facilitate its removal from a solid support. For example, an enzyme can be used to remove a nucleic acid from a substrate. For example, a nucleic acid can be removed from a substrate through a restriction endonucelase digestion. For example, treatment of a nucleic acid containing a dUTP or ddUTP with uracil-d-glycosylase (UDG) can be used to remove a nucleic acid from a substrate. For example, a nucleic acid can be removed from a substrate using an enyme that performs nucleotide excision, such as a base excision repair enzyme, such as an apurinic/apyrimidinic (AP) endonuclease. In some embodiments, a nucleic acid can be removed from a substrate using a photocleavable group and light. In some embodiments, a cleavable linker can be used to remove a nucleic acid from the substrate. For example, the cleavable linker can comprise at least one of biotin/avidin, biotin/streptavidin, biotin/neutravidin, Ig-protein A, a photo-labile linker, acid or base labile linker group, or an aptamer.

When the probes are gene-specific, the molecules can hybridize to the probes and be reverse transcribed and/or amplified. In some embodiments, after the nucleic acid has been synthesized (e.g., reverse transcribed), it may be amplified. Amplification may be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification may add sequencing adaptors to the nucleic acid.

Amplification can be performed on the substrate, for example, with bridge amplification. cDNAs can be homopolymer tailed in order to generate a compatible end for bridge amplification using oligo dT probes on the substrate. In bridge amplification, the primer that is complementary to the 3′ end of the template nucleic acid can be the first primer of each pair that is covalently attached to the solid particle. When a sample containing the template nucleic acid is contacted with the particle and a single thermal cycle is performed, the template molecule can be annealed to the first primer and the first primer is elongated in the forward direction by addition of nucleotides to form a duplex molecule consisting of the template molecule and a newly formed DNA strand that is complementary to the template. In the heating step of the next cycle, the duplex molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing stage of the annealing and elongation step that follows, the complementary strand can hybridize to the second primer, which is complementary to a segment of the complementary strand at a location removed from the first primer. This hybridization can cause the complementary strand to form a bridge between the first and second primers secured to the first primer by a covalent bond and to the second primer by hybridization. In the elongation stage, the second primer can be elongated in the reverse direction by the addition of nucleotides in the same reaction mixture, thereby converting the bridge to a double-stranded bridge. The next cycle then begins, and the double-stranded bridge can be denatured to yield two single-stranded nucleic acid molecules, each having one end attached to the particle surface via the first and second primers, respectively, with the other end of each unattached. In the annealing and elongation step of this second cycle, each strand can hybridize to a further complementary primer, previously unused, on the same particle, to form new single-strand bridges. The two previously unused primers that are now hybridized elongate to convert the two new bridges to double-strand bridges.

The amplification reactions may comprise amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids.

Amplification of the labeled nucleic acids can comprise PCR-based methods or non-PCR based methods. Amplification of the labeled nucleic acids may comprise exponential amplification of the labeled nucleic acids. Amplification of the labeled nucleic acids may comprise linear amplification of the labeled nucleic acids. Amplification can be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, suppression PCR, semi-suppressive PCR and assembly PCR.

In some instances, amplification of the labeled nucleic acids comprises non-PCR based methods. Examples of non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, a ligase chain reaction (LCR), a Qβ replicase (Qβ), use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using a restriction endonuclease, an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to the extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5′ exonuclease activity, rolling circle amplification, and/or ramification extension amplification (RAM).

In some instances, the methods disclosed herein further comprise conducting a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon may be double-stranded molecule. The double-stranded molecule may comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or a RNA molecule hybridized to a DNA molecule. One or both of the strands of the double-stranded molecule may comprise a sample tag or molecular identifier label. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present invention may comprise synthetic or altered nucleic acids.

In some instances, the method comprises repeatedly amplifying the labeled nucleic acid to produce multiple amplicons. The methods disclosed herein may comprise conducting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the method comprises conducting at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

Amplification can further comprise adding one or more control nucleic acids to one or more samples comprising a plurality of nucleic acids. Amplification can further comprise adding one or more control nucleic acids to a plurality of nucleic acids. The control nucleic acids may comprise a control label.

Amplification can comprise use of one or more non-natural nucleotides. Non-natural nucleotides may comprise photolabile and/or triggerable nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Non-natural nucleotides may be added to one or more cycles of an amplification reaction. The addition of the non-natural nucleotides may be used to identify products as specific cycles or time points in the amplification reaction.

Conducting the one or more amplification reactions may comprise the use of one or more primers. The one or more primers can comprise one or more oligonucleotides. The one or more oligonucleotides can comprise at least about 7-9 nucleotides. The one or more oligonucleotides may comprise less than 12-15 nucleotides. The one or more primers may anneal to at least a portion of the plurality of labeled nucleic acids. The one or more primers may anneal to the 3′ end and/or 5′ end of the plurality of labeled nucleic acids. The one or more primers may anneal to an internal region of the plurality of labeled nucleic acids. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3′ ends the plurality of labeled nucleic acids. The one or more primers may comprise a fixed panel of primers. The one or more primers may comprise at least one or more custom primers. The one or more primers may comprise at least one or more control primers. The one or more primers can comprise at least one or more housekeeping gene primers. The one or more primers may comprise a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to the first sample tag, the second sample tag, the molecular identifier label, the nucleic acid or a product thereof. The one or more primers may comprise a universal primer and a custom primer. The custom primer may be designed to amplify one or more target nucleic acids. The target nucleic acids may comprise a subset of the total nucleic acids in one or more samples. In some instances, the primers are the probes attached to the array of the disclosure.

Detection

One or more probes that are target-specific may be used to detect one or more targets on the substrate. In some embodiments, the probes may be fluorescently labeled. An image of the hybridized probes may be generated by, for example, fluorescent imaging. In some embodiments, the probes may be removed and a different set of probes are used to detect a different set of targets.

The targets (e.g., molecules, amplified molecules) can be detected, for example, using detection probes (e.g., fluorescent probes). The array can be hybridized with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more detection probes. The array can be hybridized with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more detection probes. In some embodiments, the array is hybridized with 4 detection probes.

The detection probes can comprise a sequence complementary to a sequence of a gene of interest. The length of the detection probe can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The length of the detection probe can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The detection probes can comprise a sequence that is perfectly complementary to a sequence in a gene of interest (e.g., target). The detection probes can comprise a sequence that is imperfectly complementary to a sequence in a gene of interest (e.g., target). The detection probes can comprise a sequence with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches to the sequence of the gene of interest. The detection probes can comprise a sequence with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches to the sequence of the gene of interest.

The detection probes can comprise a detectable label. Exemplary detectable labels can comprise a fluorophore, chromophore, small molecule, nanoparticle, hapten, enzyme, antibody, and magnetic property, or any combination thereof.

Hybridized probes can be imaged. The image can be used to determine the relative expression level of the genes of interest based on the intensity of the detectable signal (e.g., fluorescent signal). Scanning laser fluorescence microscopes or readers can be used to acquire digital images of the emitted light from substrate (e.g., microarray). A focused light source (usually a laser) can be scanned across the hybridized substrate causing the hybridized areas to emit an optical signal, such as fluorescence. The fluorophore-specific fluorescence data can be collected and measured during the scanning operation, and then an image of the substrate can be reconstructed via appropriate algorithms, software and computer hardware. The expected or intended locations of probe nucleic acid features can then be combined with the fluorescence intensities measured at those locations, to yield the data that is then used to determine gene expression levels or nucleic acid sequence of the target samples. The process of collecting data from expected probe locations can be referred to as “feature extraction”. The digital images can be comprised of several thousand to hundreds of millions of pixels that typically range in size from 5 to 50 microns. Each pixel in the digital image can be represented by a 16 bit integer, allowing for 65,535 different grayscale values. The reader can sequentially acquire the pixels from the scanned substrate and writes them into an image file which can be stored on a computer hard drive. The substrates can contain several different fluorescently tagged probe DNA samples at each spot location. The scanner repeatedly scans the entire substrate with a laser of the appropriate wavelength to excite each of the probe DNA samples and store them in their separate image files. The image files are analyzed and subsequently viewed with the aid of a programmed computer.

The substrate can be imaged with a confocal laser scanner. The scanner can scan the substrate slide to produce one image for each dye used by sequentially scanning the with a laser of a proper wavelength for the particular dye. Each dye can have a known excitation spectra and a known emission spectra. The scanner can include a beam splitter which reflects a laser beam towards an objective lens which, in turn, focuses the beam at the surface of slide to cause fluorescence spherical emission. A portion of the emission can travel back through the lens and the beam splitter. After traveling through the beam splitter, the fluorescence beam can be reflected by a mirror, travels through an emission filter, a focusing detector lens and a central pinhole.

Correlation Between Probing and Imaging Data

By correlating the image showing the locations of the target with an image of the sample, the spatial barcode of the target may be generated. The data from the substrate scan can be correlated to the image of the unlysed sample on the substrate. The data can be overlayed thereby generating a map. A map of the location of targets from a sample can be constructed using information generated using the methods described herein. The map can be used to locate a physical location of a target. The map can be used to identify the location of multiple targets. The multiple targets can be the same species of target, or the multiple targets can be multiple different targets. For example a map of a brain can be constructed to show the amount and location of multiple targets.

The map can be generated from data from a single sample. The map can be constructed using data from multiple samples, thereby generating a combined map. The map can be constructed with data from tens, hundreds, and/or thousands of samples. A map constructed from multiple samples can show a distribution of targets associated with regions common to the multiple samples. For example, replicated assays can be displayed on the same map. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more replicates can be displayed (e.g., overlaid) on the same map. At most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more replicates can be displayed (e.g., overlaid) on the same map. The spatial distribution and number of targets can be represented by a variety of statistics.

Combining data from multiple samples can increase the locational resolution of the combined map. The orientation of multiple samples can be registered by common landmarks and/or x-y positions on the array, wherein the individual locational measurements across samples are at least in part non-contiguous. Multiplexing the above approach will allow for high resolution maps of target nucleic acids in a sample.

The data analysis and correlation can be useful for determining the presence and/or absence of a specific cell type (e.g., rare cell, cancer cell). The data correlation can be useful for determining the relative ratios of target nucleic acids in distinct locations either within a cell, or within a sample.

The methods and compositions disclosed herein can be companion diagnostics for a medical professional (e.g., a pathologist) wherein a subject can be diagnosed by visually looking at a pathology image and correlating the image to genetic expression (e.g., identification of expression of oncogenes). The methods and compositions can be useful for identifying a cell from a population of cells, and determining the genetic heterogeneity of the cells within a sample. The methods and compositions can be useful for determining the genotype of a sample.

The disclosure provides for methods for making replicates of substrates. The substrates can be reprobed with different probes for different genes of interest, or to selectively choose specific genes. For example, a sample can be placed on a substrate comprising a plurality of oligo(dT) probes. mRNAs can hybridize to the probes. Replicate substrates comprising oligo(dT) probes can be contacted to the initial slide and make replicates of the mRNAs. Replicate substrates comprising RNA gene-specific probes can be contacted to the initial slide to make a replicate.

The mRNA can be reverse transcribed into cDNA. The cDNA can be homopolymer tailed and/or amplified (e.g., via bridge amplification). The array can be contacted with a replicate array. The replicate array can comprise gene-specific probes that can bind to the cDNAs of interest. The replicate array can comprise polyA probes that can bind to cDNAs with a polyadenylation sequence.

The number of replicates that can be made can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The number of replicates that can be made can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more.

In some embodiments, the initial substrate comprises a plurality of gene-specific probes and the replicate substrate comprises the same gene-specific probes, or different probes that correspond to the same genes as the gene-specific probes.

Imaging

The sample contacted to the substrate can be analyzed (e.g., with immunohistochemistry, staining and/or imaging). Exemplary methods of immunohistochemistry can comprise a step of reacting a labeled probe biological substance obtained by introducing a label into a substance capable of recognizing a biological substance to be detected to a tissue section, to visualize the biological substance to be detected present on the tissue section via a specific binding reaction between the biological substances.

For histology specimens, the tissue pieces can be fixed in a suitable fixative, typically formalin, and embedded in melted paraffin wax. The wax block can be cut on a microtome to yield a thin slice of paraffin containing the tissue. The specimen slice can be applied to a substrate, air dried, and heated to cause the specimen to adhere to the glass slide. Residual paraffin can be dissolved with a suitable solvent, typically xylene, toluene, or others. These so-called deparaffinizing solvents can be removed with a washing-dehydrating type reagent prior to staining. Slices can be prepared from frozen specimens, fixed briefly in 10% formalin, then infused with dehydrating reagent. The dehydrating reagent can be removed prior to staining with an aqueous stain.

In some embodiments, the Papanicolaou staining technique can be used (e.g., a progressive stain and/or hematoxylineosin [H&E], i.e., a regressive stain). HE (hematoxylin-eosin) stain uses hematoxylin and eosin as a dye. Hematoxylin is a blue-violet dye, and has a property of staining basophilic tissues such as cell nuclei, bone tissues, part of cartilage tissues, and serous components. Eosin is a red to pink dye, and has a property of staining eosinophilic tissues such as cytoplasm, connective tissues of the softtissue, red blood cells, fibrin, and endocrine granules.

Immunohistochemistry (IHC) can be referred to as “immunological staining” due to the process of color development for visualizing an antigen-antibody reaction which is otherwise invisible (hereinafter, the term “immunohistochemical staining” can be used for immunohistochemistry). Lectin staining is a technique that can use a property of lectin of binding to a specific sugar chain in a non-immunological and specific manner in order to detect a sugar chain in a tissue specimen using lectin.

HE staining, immunohistochemistry and lectin staining can be used for detecting a location of, for example, cancer cells in a cell specimen. For example, when it is desired to confirm a location of cancer cells in a cell specimen, a pathologist, in order to determine the presence or absence of cancer cells in the cell specimen, can prepare tissue sections and place them on a substrate of the disclosure. The section on the array can be subjected to HE staining, imaging, or any immunohistochemical analysis in order to obtain its morphological information and/or any other identifying features (such as presence or absence of rare cells). The sample can be lysed and the presence or absence of nucleic acid molecules can be determined using the methods of the disclosure. The nucleic acid information can be compared (e.g., spatially compared) to the image, thereby indicating the spatial location of nucleic acids in a sample.

In some embodiments, the tissue is stained with a staining enhancer (e.g., a chemical penetrant enhancer). Examples of tissue chemical penetrant enhancers that facilitate penetration of the stain into the tissue include, but are not limited to, polyethylene glycol (PEG), surfactants such as polyoxyethylenesorbitans, polyoxyethylene ethers (polyoxyethylenesorbitan monolaurate (Tween 20) and other Tween derivatives, polyoxyethylene 23 lauryl ether (Brij 35), Triton X-100, Brij 35, Nonidet P-40, detergent-like substances such as lysolecithins, saponins, non-ionic detergents such as TRITON® X-100, etc., aprotic solvents such as dimethyl sulfoxide (DMSO), ethers such as tetrahydrofuran, dioxane, etc.; esters such as ethyl acetate, butyl acetate, isopropyl acetate; hydrocarbons such as toluene, chlorinated solvents such as dichloromethane, dichloroethane, chlorobenzene, etc.; ketones such as acetone, nitriles such as acetonitrile, and/or other agents that increase cell membrane permeability.

In some embodiments, a composition is provided that facilitates staining of a mammalian tissue sample. The composition can comprise a stain, such as hematoxylin, or hematoxylin and eosin-Y, at least one tissue chemical penetrant enhancer, such as a surfactant, an aprotic solvent, and/or PEG, or any combination thereof.

In some embodiments, the sample is imaged (e.g., either before or after IHC or without IHC). Imaging can comprise microscopy such as bright field imaging, oblique illumination, dark field imaging, dispersion staining, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence, confocal, electron microscopy, transmission electron microscopy, scanning electron microscopy, and single plane illumination, or any combination thereof. Imaging can comprise the use of a negative stain (e.g., nigrosin, ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide). Imaging can comprise the use of heavy metals (e.g., gold, osmium) that can scatter electrons.

Imaging can comprise imaging a portion of the sample (e.g., slide/array). Imaging can comprise imaging at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the sample. Imaging can comprise imaging at most 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the sample. Imaging can be done in discrete steps (e.g., the image may not need to be contiguous). Imaging can comprise taking at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different images. Imaging can comprise taking at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different images.

FIG. 9 illustrates an exemplary embodiment of the homopolymer tailing method of the disclosure The disclosure provides for a substrate 910 comprising a plurality of probes 905 attached to the surface of the substrate. The substrate 910 can be a microarray. The plurality of probes 905 can comprise an oligo(dT). The plurality of probes 905 can comprise a gene-specific sequence. The plurality of probes 105 can comprise a stochastic barcode. A sample (e.g., cells) 915 can be placed and/or grown on the substrate 910. The substrate comprising the sample can be analyzed 920, for example by imaging and/or immunohistochemistry. The sample 915 can be lysed 925 on the substrate 910. The nucleic acids 930 from the sample 915 can associate (e.g., hybridize) with the plurality of probes 905 on the substrate 910. In some embodiments, the nucleic acids 930 can be reverse transcribed, homopolymer tailed, and/or amplified (e.g., with bridge amplification). The amplified nucleic acids can be interrogated 935 with detection probes 940 (e.g., fluorescent probes). The detection probes 940 can be gene-specific probes. The location of binding of the detection probes 940 on the substrate 910 can be correlated with the image of the substrate, thereby producing a map that indicates the spatial location of nucleic acids in the sample.

In some embodiments, the methods can comprise making 945 a replicate 946 of the original substrate 910. The replicate substrate 946 can comprise a plurality of probes 931. The plurality of probes 931 can be the same as the plurality of probes 905 on the original substrate 910. The plurality of probes 931 can be different than the plurality of probes 905 on the original substrate 910. For example, the plurality of probes 905 can be oligo(dT) probes and the plurality of probes 931 on the replicate substrate 946 can be gene-specific probes. The replicate substrate can be processed like the original substrate, such as with interrogation by detection (e.g., fluorescent) probes.

Samples Cells

A sample for use in the method of the disclosure can comprise one or more cells. A sample can refer to one or more cells. In some embodiments, the cells are cancer cells excised from a cancerous tissue, for example, breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer, melanoma and non-melanoma skin cancers, and the like. In some instances, the cells are derived from a cancer but collected from a bodily fluid (e.g. circulating tumor cells). Non-limiting examples of cancers may include, adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma, and fibrosarcoma.

In some embodiments, the cells are cells that have been infected with virus and contain viral oligonucleotides. In some embodiments, the viral infection may be caused by a virus selected from the group consisting of double-stranded DNA viruses (e.g. adenoviruses, herpes viruses, pox viruses), single-stranded (+ strand or “sense”) DNA viruses (e.g. parvoviruses), double-stranded RNA viruses (e.g. reoviruses), single-stranded (+ strand or sense) RNA viruses (e.g. picornaviruses, togaviruses), single-stranded (− strand or antisense) RNA viruses (e.g. orthomyxoviruses, rhabdoviruses), single-stranded ((+ strand or sense) RNA viruses with a DNA intermediate in their life-cycle) RNA-RT viruses (e.g. retroviruses), and double-stranded DNA-RT viruses (e.g. hepadnaviruses). Exemplary viruses can include, but are not limited to, SARS, HIV, coronaviruses, Ebola, Malaria, Dengue, Hepatitis C, Hepatitis B, and Influenza.

In some embodiments, the cells are bacteria. These may include either gram-positive or gram-negative bacteria. Examples of bacteria that may be analyzed using the disclosed methods, devices, and systems include, but are not limited to, Actinomedurae, Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium, Enterococcus faecalis, Listeria monocytogenes, Nocardia, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epiderm, Streptococcus mutans, Streptococcus pneumoniae and the like. Gram negative bacteria include, but are not limited to, Afipia felis, Bacteroides, Bartonella bacilliformis, Bortadella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella, Calymmatobacterium granulomatis, Campylobacter, Escherichia coli, Francisella tularensis, Gardnerella vaginalis, Haemophilius aegyptius, Haemophilius ducreyi, Haemophilius influenziae, Heliobacter pylori, Legionella pneumophila, Leptospira interrogans, Neisseria meningitidia, Porphyromonas gingivalis, Providencia sturti, Pseudomonas aeruginosa, Salmonella enteridis, Salmonella typhi, Serratia marcescens, Shigella boydii, Streptobacillus moniliformis, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis and the like. Other bacteria may include Myobacterium avium, Myobacterium leprae, Myobacterium tuberculosis, Bartonella henseiae, Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasma pneumoniae, Rickettsia akari, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia tsutsugamushi, Rickettsia typhi, Ureaplasma urealyticum, Diplococcus pneumoniae, Ehrlichia chafensis, Enterococcus faecium, Meningococci and the like.

In some embodiments, the cells are fungi. Non-limiting examples of fungi that may be analyzed using the disclosed methods, devices, and systems include, but are not limited to, Aspergilli, Candidae, Candida albicans, Coccidioides immitis, Cryptococci, and combinations thereof.

In some embodiments, the cells are protozoans or other parasites. Examples of parasites to be analyzed using the methods, devices, and systems of the present disclosure include, but are not limited to, Balantidium coli, Cryptosporidium parvum, Cyclospora cayatanensis, Encephalitozoa, Entamoeba histolytica, Enterocytozoon bieneusi, Giardia lamblia, Leishmaniae, Plasmodii, Toxoplasma gondii, Trypanosomae, trapezoidal amoeba, worms (e.g., helminthes), particularly parasitic worms including, but not limited to, Nematoda (roundworms, e.g., whipworms, hookworms, pinworms, ascarids, filarids and the like), Cestoda (e.g., tapeworms).

As used herein, the term “cell” can refer to one or more cells. In some embodiments, the cells are normal cells, for example, human cells in different stages of development, or human cells from different organs or tissue types (e.g. white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, small intestine). In some embodiments, the cells may be undifferentiated human stem cells, or human stem cells that have been induced to differentiate. In some embodiments, the cells may be fetal human cells. The fetal human cells may be obtained from a mother pregnant with the fetus. In some embodiments, the cells are rare cells. A rare cell may be, for example, a circulating tumor cell (CTC), circulating epithelial cell, circulating endothelial cell, circulating endometrial cell, circulating stem cell, stem cell, undifferentiated stem cell, cancer stem cell, bone marrow cell, progenitor cell, foam cell, mesenchymal cell, trophoblast, immune system cell (host or graft), cellular fragment, cellular organelle (e.g. mitochondria or nuclei), pathogen infected cell, and the like.

In some embodiments, the cells are non-human cells, for example, other types of mammalian cells (e.g. mouse, rat, pig, dog, cow, or horse). In some embodiments, the cells are other types of animal or plant cells. In other embodiments, the cells may be any prokaryotic or eukaryotic cells.

In some embodiments, a first cell sample is obtained from a person not having a disease or condition, and a second cell sample is obtained from a person having the disease or condition. In some embodiments, the persons are different. In some embodiments, the persons are the same but cell samples are taken at different time points. In some embodiments, the persons are patients, and the cell samples are patient samples. The disease or condition can be a cancer, a bacterial infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a parasitic disease, a genetic disorder, or any combination thereof.

In some embodiments, cells suitable for use in the presently disclosed methods range in size from about 2 micrometers to about 100 micrometers in diameter. In some embodiments, the cells have diameters of at least 2 micrometers, at least 5 micrometers, at least 10 micrometers, at least 15 micrometers, at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, at least 60 micrometers, at least 70 micrometers, at least 80 micrometers, at least 90 micrometers, or at least 100 micrometers. In some embodiments, the cells have diameters of at most 100 micrometers, at most 90 micrometers, at most 80 micrometers, at most 70 micrometers, at most 60 micrometers, at most 50 micrometers, at most 40 micrometers, at most 30 micrometers, at most 20 micrometers, at most 15 micrometers, at most 10 micrometers, at most 5 micrometers, or at most 2 micrometers. The cells can have a diameter of any value within a range, for example from about 5 micrometers to about 85 micrometers. In some embodiments, the cells have diameters of about 10 micrometers.

In some embodiments the cells are sorted prior to associating a cell with a bead and/or in a microwell. For example the cells can be sorted by fluorescence-activated cell sorting or magnetic-activated cell sorting, or e.g., by flow cytometry. The cells can be filtered by size. In some instances a retentate contains the cells to be associated with the bead. In some instances the flow through contains the cells to be associated with the bead.

The sample can be nucleic acids. The sample can comprise nucleic acids. The sample can be a single cell. When the sample is a single cell, the cell may be lysed to release the nucleic acids from the single cell. The sample can be multiple cells. Samples in different wells can come from the same subject. Samples in different wells can come from different subjects. Samples in different wells can come from different tissues (e.g., brain, heart, lung, kidney, spleen). Samples in different wells can come from the same tissue.

Diffusion Across a Substrate

When a sample (e.g., cell) is stochastically barcoded and/or combinatorially barcoded according to the methods of the disclosure, the cell may be lysed. Lysis of a cell can result in the diffusion of the contents of the lysis (e.g., cell contents) away from the initial location of lysis. In other words, the lysis contents can move into a larger surface area than the surface area taken up by the cell.

Diffusion of sample lysis mixture (e.g., comprising targets) can be modulated by various parameters including, but not limited to, viscosity of the lysis mixture, temperature of the lysis mixture, the size of the targets, the size of physical barriers in a substrate, the concentration of the lysis mixture, and the like. For example, the temperature of the lysis reaction can be performed at a temperature of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 C or more. The temperature of the lysis reaction can be performed at a temperature of at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 C or more. The viscosity of the lysis mixture can be altered by, for example, adding thickening reagents (e.g., glycerol, beads) to slow the rate of diffusion. The viscosity of the lysis mixture can be altered by, for example, adding thinning reagents (e.g., water) to increase the rate of diffusion. A substrate can comprise physical barriers (e.g., wells, microwells, microhills) that can alter the rate of diffusion of targets from a sample. The concentration of the lysis mixture can be altered to increase or decrease the rate of diffusion of targets from a sample. The concentration of a lysis mixture can be increased or decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more fold. The concentration of a lysis mixture can be increased or decreased by at most 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more fold.

The rate of diffusion can be increased. The rate of diffusion can be decreased. The rate of diffusion of a lysis mixture can be increased or decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fold compared to an un-altered lysis mixture. The rate of diffusion of a lysis mixture can be increased or decreased by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fold compared to an un-altered lysis mixture. The rate of diffusion of a lysis mixture can be increased or decreased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% compared to an un-altered lysis mixture. The rate of diffusion of a lysis mixture can be increased or decreased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% compared to an un-altered lysis mixture.

Data Analysis and Display Software Data Analysis and Visualization of Spatial Resolution of Targets

The disclosure provides for methods for estimating the number and position of targets with stochastic barcoding and/or combinatorial barcoding and digital counting. The data obtained from the methods of the disclosure can be visualized on a map. The data obtained from the methods of the disclosure can be visualized on a map of the microwell array (e.g., such that the results of each sample can be traced to a location in the microwell array). A map of the number and location of targets from a sample can be constructed using information generated using the methods described herein. The map can be used to locate a physical location of a target. The map can be used to identify the location of multiple targets. The multiple targets can be the same species of target, or the multiple targets can be multiple different targets. For example a map of a brain can be constructed to show the digital count and location of multiple targets.

The map can be generated from data from a single sample. The map can be constructed using data from multiple samples, thereby generating a combined map. The map can be constructed with data from tens, hundreds, and/or thousands of samples. A map constructed from multiple samples can show a distribution of digital counts of targets associated with regions common to the multiple samples. For example, replicated assays can be displayed on the same map. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more replicates may be displayed (e.g., overlaid) on the same map. At most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more replicates may be displayed (e.g., overlaid) on the same map. The spatial distribution and number of targets can be represented by a variety of statistics.

In some embodiments of the instrument system, the system will comprise computer-readable media that includes code for providing data analysis for the sequence datasets generated by performing single cell, stochastic barcoding assays. Examples of data analysis functionality that may be provided by the data analysis software include, but are not limited to, (i) algorithms for decoding/demultiplexing of the sample label, cell label, molecular label, and target sequence data provided by sequencing the stochastic barcode and/or combinatorial barcode reagent library created in running the assay, (ii) algorithms for determining the number of reads per gene per cell, and the number of unique transcript molecules per gene per cell, based on the data, and creating summary tables, (iii) statistical analysis of the sequence data, e.g. for clustering of cells by gene expression data, or for predicting confidence intervals for determinations of the number of transcript molecules per gene per cell, etc., (iv) algorithms for identifying sub-populations of rare cells, for example, using principal component analysis, hierarchical clustering, k-mean clustering, self-organizing maps, neural networks etc., (v) sequence alignment capabilities for alignment of gene sequence data with known reference sequences and detection of mutation, polymorphic markers and splice variants, and (vi) automated clustering of molecular labels to compensate for amplification or sequencing errors. In some embodiments, commercially-available software may be used to perform all or a portion of the data analysis, for example, the Seven Bridges (https://www.sbgenomics.com/) software may be used to compile tables of the number of copies of one or more genes occurring in each cell for the entire collection of cells. In some embodiments, the data analysis software may include options for outputting the sequencing results in useful graphical formats, e.g. heatmaps that indicate the number of copies of one or more genes occurring in each cell of a collection of cells. In some embodiments, the data analysis software may further comprise algorithms for extracting biological meaning from the sequencing results, for example, by correlating the number of copies of one or more genes occurring in each cell of a collection of cells with a type of cell, a type of rare cell, or a cell derived from a subject having a specific disease or condition. In some embodiment, the data analysis software may further comprise algorithms for comparing populations of cells across different biological samples.

In some embodiments all of the data analysis functionality may be packaged within a single software package. In some embodiments, the complete set of data analysis capabilities may comprise a suite of software packages. In some embodiments, the data analysis software may be a standalone package that is made available to users independently of the assay instrument system. In some embodiments, the software may be web-based, and may allow users to share data.

System Processors and Networks

In general, the computer or processor included in the presently disclosed instrument systems, may be further understood as a logical apparatus that can read instructions from media or a network port, which can optionally be connected to server having fixed media. The system can include a CPU, disk drives, optional input devices such as keyboard or mouse and optional monitor. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception or review by a party.

An exemplary embodiment of a first example architecture of a computer system can be used in connection with example embodiments of the present disclosure. The example computer system can include a processor for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some embodiments, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, or personal data assistant devices.

A high speed cache can be connected to, or incorporated in, the processor to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor. The processor can be connected to a north bridge by a processor bus. The north bridge is connected to random access memory (RAM) by a memory busand manages access to the RAM by the processor. The north bridge can also be connected to a south bridge by a chipset bus. The south bridge is, in turn, connected to a peripheral bus. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip.

The system can include an accelerator card attached to the peripheral bus. The accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing. For example, an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

Software and data can be stored in external storage and can be loaded into RAM or cache for use by the processor. The system includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example embodiments of the present invention.

In this example, system also includes network interface cards (NICs) and connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.

An exemplary diagram of a network can comprise a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS). In example embodiments, systems can manage data storage and optimize data access for data stored in Network Attached Storage (NAS). A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems, and cell phone and personal data assistant systems. Computer systems, and cell phone and personal data assistant systems can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS). A wide variety of other computer architectures and systems can be used in conjunction with the various embodiments of the present invention. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface.

In some example embodiments, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other embodiments, some or all of the processors can use a shared virtual address memory space.

An exemplary a block diagram of a multiprocessor computer system can comprise a shared virtual address memory space in accordance with an example embodiment. The system can include a plurality of processors that can access a shared memory subsystem. The system can incorporate a plurality of programmable hardware memory algorithm processors (MAPs) in the memory subsystem. Each MAP can comprise a memory and one or more field programmable gate arrays (FPGAs). The MAP can provide a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example embodiments. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory, allowing it to execute tasks independently of, and asynchronously from the respective microprocessor. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example embodiments, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some embodiments, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.

In example embodiments, the computer subsystem of the present disclosure can be implemented using software modules executing on any of the above or other computer architectures and systems. In other embodiments, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card.

Kits

Disclosed herein are kits for performing single cell, stochastic barcoding and/or combinatorial barcoding assays. The kit can comprise any composition or mixtures of compositions of the disclosure (e.g., any combinatorial barcode reagent of any type and any number). The kit can comprise one or more combinatorial barcode reagents of the disclosure. The kit can comprise one or more stochastic barcodes of the disclosure. The kit can comprise one or more substrates (e.g., microwell array), either as a free-standing substrate (or chip) comprising one or more microwell arrays, or packaged within one or more flow-cells or cartridges. The one or more substrates of the kit can comprise the combinatorial barcode reagents and/or stochastic barcode reagents pre-loaded in the wells. In some instances, the stochastic barcode reagents may be pre-loaded in the wells of the substrate and the kit comprises combinatorial barcode reagents for user addition to the wells.

In some instances, a kit can comprise a set of combinatorial barcode reagents. The second of combinatorial barcode reagents can include one combinatorial barcode reagent that comprises a target-specific region (e.g., either to the sense or anti-sense strand), and other combinatorial barcode reagents that may not comprise a target-specific region, but may comprise linkers to link all the combinatorial barcode reagents together (e.g., such that the combinatorial barcode reagents are linked together on one end of a target nucleic acid (See FIG. 2C)).

The kits can comprise one or more solid support suspensions, wherein the individual solid supports within a suspension comprise a plurality of attached stochastic barcodes of the disclosure. The kits can comprise stochastic barcodes that may not be attached to a solid support. The kit can comprise a sealant. In some embodiments, the kit may further comprise a mechanical fixture for mounting a free-standing substrate in order to create reaction wells that facilitate the pipetting of samples and reagents into the substrate.

The kit can further comprise reagents, e.g. lysis buffers, rinse buffers, or hybridization buffers, for performing the stochastic barcoding assay. The kit can further comprise reagents (e.g. enzymes, primers, dNTPs, NTPs, RNAse inhibitors, or buffers) for performing nucleic acid extension reactions, for example, reverse transcription reactions and primer extension reactions. The kit can further comprise reagents (e.g. enzymes, universal primers, sequencing primers, target-specific primers, or buffers) for performing amplification reactions to prepare sequencing libraries. The kit can comprise reagents for homopolymer tailing of molecules (e.g., a terminal transferase enzyme, and dNTPs). The kit can comprise reagents for, for example, any enzymatic cleavage of the disclosure (e.g., ExoI nuclease, restriction enzyme). The reagents can be pre-loaded in wells of a substrate (e.g., with combinatorial barcode reagents).

The kit can comprise sequencing library amplification primers. The kit may comprise a second strand synthesis primer of the disclosure. The kit can comprise any primers of the disclosure (e.g., gene-specific primers, random multimers, sequencing primers, and universal primers).

The kit can comprise one or more molds, for example, molds comprising an array of micropillars, for casting substrates (e.g., microwell arrays), and one or more solid supports (e.g., bead), wherein the individual beads within a suspension comprise a plurality of attached stochastic barcodes of the disclosure. The kit may further comprise a material for use in casting substrates (e.g. agarose, a hydrogel, PDMS, optical adhesive. and the like).

The kit can comprise one or more substrates that are pre-loaded with solid supports comprising a plurality of attached stochastic barcodes of the disclosure. In some instances, there may be one solid support per microwell of the substrate. In some embodiments, the plurality of stochastic barcodes may be attached directly to a surface of the substrate, rather than to a solid support. In any of these embodiments, the one or more microwell arrays may be provided in the form of free-standing substrates (or chips), or they may be packed in flow-cells or cartridges.

In some embodiments, the kit can comprise one or more cartridges that incorporate one or more substrates. In some embodiments, the one or more cartridges further comprise one or more pre-loaded solid supports, wherein the individual solid supports within a suspension comprise a plurality of attached stochastic barcodes of the disclosure. In some embodiments, the beads is pre-distributed into the one or more microwell arrays of the cartridge. In some embodiments, the beads, in the form of suspensions, can be pre-loaded and stored within reagent wells of the cartridge. In some embodiments, the one or more cartridges is further comprise other assay reagents that are pre-loaded and stored within reagent reservoirs of the cartridges.

Kits can generally include instructions for carrying out one or more of the methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the disclosure. Such media can include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), RF tags, and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.

Devices Flow Cells

The microwell array substrate can be packaged within a flow cell that provides for convenient interfacing with the rest of the fluid handling system and facilitates the exchange of fluids, e.g. cell and solid support suspensions, lysis buffers, rinse buffers, etc., that are delivered to the microwell array and/or emulsion droplet. Design features can include: (i) one or more inlet ports for introducing cell samples, solid support suspensions, or other assay reagents, (ii) one or more microwell array chambers designed to provide for uniform filling and efficient fluid-exchange while minimizing back eddies or dead zones, and (iii) one or more outlet ports for delivery of fluids to a sample collection point or a waste reservoir. The design of the flow cell may include a plurality of microarray chambers that interface with a plurality of microwell arrays such that one or more different cell samples may be processed in parallel. The design of the flow cell may further include features for creating uniform flow velocity profiles, i.e. “plug flow”, across the width of the array chamber to provide for more uniform delivery of cells and beads to the microwells, for example, by using a porous barrier located near the chamber inlet and upstream of the microwell array as a “flow diffuser”, or by dividing each array chamber into several subsections that collectively cover the same total array area, but through which the divided inlet fluid stream flows in parallel. In some embodiments, the flow cell may enclose or incorporate more than one microwell array substrate. In some embodiments, the integrated microwell array/flow cell assembly may constitute a fixed component of the system. In some embodiments, the microwell array/flow cell assembly may be removable from the instrument.

In general, the dimensions of fluid channels and the array chamber(s) in flow cell designs will be optimized to (i) provide uniform delivery of cells and beads to the microwell array, and (ii) to minimize sample and reagent consumption. In some embodiments, the width of fluid channels will be between 50 um and 20 mm. In other embodiments, the width of fluid channels may be at least 50 um, at least 100 um, at least 200 um, at least 300 um, at least 400 um, at least 500 um, at least 750 um, at least 1 mm, at least 2.5 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, or at least 150 mm. In yet other embodiments, the width of fluid channels may be at most 150 mm, at most 100 mm, at most 50 mm, at most 20 mm, at most 10 mm, at most 5 mm, at most 2.5 mm, at most 1 mm, at most 750 um, at most 500 um, at most 400 um, at most 300 um, at most 200 um, at most 100 um, or at most 50 um. In one embodiment, the width of fluid channels is about 2 mm. The width of the fluid channels may fall within any range bounded by any of these values (e.g. from about 250 um to about 3 mm).

In some embodiments, the depth of the fluid channels can be between 50 um and 2 mm. In other embodiments, the depth of fluid channels can be at least 50 um, at least 100 um, at least 200 um, at least 300 um, at least 400 um, at least 500 um, at least 750 um, at least 1 mm, at least 1.25 mm, at least 1.5 mm, at least 1.75 mm, or at least 2 mm. In yet other embodiments, the depth of fluid channels can be at most 2 mm, at most 1.75 mm, at most 1.5 mm, at most 1.25 mm, at most 1 mm, at most 750 um, at most 500 um, at most 400 um, at most 300 um, at most 200 um, at most 100 um, or at most 50 um. In one embodiment, the depth of the fluid channels is about 1 mm. The depth of the fluid channels may fall within any range bounded by any of these values (e.g. from about 800 um to about 1 mm).

Flow cells may be fabricated using a variety of techniques and materials known to those of skill in the art. For example, the flow cell can be fabricated as a separate part and subsequently either mechanically clamped or permanently bonded to the microwell array substrate. Examples of suitable fabrication techniques include conventional machining, CNC machining, injection molding, 3D printing, alignment and lamination of one or more layers of laser or die-cut polymer films, or any of a number of microfabrication techniques such as photolithography and wet chemical etching, dry etching, deep reactive ion etching, or laser micromachining. Once the flow cell part has been fabricated it may be attached to the microwell array substrate mechanically, e.g. by clamping it against the microwell array substrate (with or without the use of a gasket), or it may be bonded directly to the microwell array substrate using any of a variety of techniques (depending on the choice of materials used) known to those of skill in the art, for example, through the use of anodic bonding, thermal bonding, or any of a variety of adhesives or adhesive films, including epoxy-based, acrylic-based, silicone-based, UV curable, polyurethane-based, or cyanoacrylate-based adhesives.

Flow cells can be fabricated using a variety of materials known to those of skill in the art. In general, the choice of material used will depend on the choice of fabrication technique used, and vice versa. Examples of suitable materials include, but are not limited to, silicon, fused-silica, glass, any of a variety of polymers, e.g. polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxy resins, metals (e.g. aluminum, stainless steel, copper, nickel, chromium, and titanium), a non-stick material such as teflon (PTFE), or a combination of these materials.

Cartridges

In some embodiments of the system, the microwell array, with or without an attached flow cell, can be packaged within a consumable cartridge that interfaces with the instrument system. Design features of cartridges can include (i) one or more inlet ports for creating fluid connections with the instrument or manually introducing cell samples, bead suspensions, or other assay reagents into the cartridge, (ii) one or more bypass channels, i.e. for self-metering of cell samples and bead suspensions, to avoid overfilling or back flow, (iii) one or more integrated microwell array/flow cell assemblies, or one or more chambers within which the microarray substrate(s) are positioned, (iv) integrated miniature pumps or other fluid actuation mechanisms for controlling fluid flow through the device, (v) integrated miniature valves (or other containment mechanisms) for compartmentalizing pre-loaded reagents (for example, bead suspensions, combinatorial barcode reagents, stochastic barcodes) or controlling fluid flow through the device, (vi) one or more vents for providing an escape path for trapped air, (vii) one or more sample and reagent waste reservoirs, (viii) one or more outlet ports for creating fluid connections with the instrument or providing a processed sample collection point, (ix) mechanical interface features for reproducibly positioning the removable, consumable cartridge with respect to the instrument system, and for providing access so that external magnets can be brought into close proximity with the microwell array, (x) integrated temperature control components or a thermal interface for providing good thermal contact with the instrument system, and (xi) optical interface features, e.g. a transparent window, for use in optical interrogation of the microwell array.

The cartridge can be designed to process more than one sample in parallel. The cartridge may further comprise one or more removable sample collection chamber(s) that are suitable for interfacing with stand-alone PCR thermal cyclers or sequencing instruments. The cartridge itself can be suitable for interfacing with stand-alone PCR thermal cyclers or sequencing instruments. The term “cartridge” as used in this disclosure can be meant to include any assembly of parts which contains the sample and beads during performance of the assay.

The cartridge can further comprise components that are designed to create physical or chemical barriers that prevent diffusion of (or increase path lengths and diffusion times for) large molecules in order to minimize cross-contamination between microwells. Examples of such barriers can include, but are not limited to, a pattern of serpentine channels used for delivery of cells and solid supports (e.g., beads) to the microwell array, a retractable platen or deformable membrane that is pressed into contact with the surface of the microwell array substrate during lysis or incubation steps, the use of larger beads, e.g. Sephadex beads as described previously, to block the openings of the microwells, or the release of an immiscible, hydrophobic fluid from a reservoir within the cartridge during lysis or incubation steps, to effectively separate and compartmentalize each microwell in the array.

The dimensions of fluid channels and the array chamber(s) in cartridge designs can be optimized to (i) provide uniform delivery of cells and beads to the microwell array, and (ii) to minimize sample and reagent consumption. The width of fluid channels can be between 50 micrometers and 20 mm. In other embodiments, the width of fluid channels may be at least 50 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, at least 400 micrometers, at least 500 micrometers, at least 750 micrometers, at least 1 mm, at least 2.5 mm, at least 5 mm, at least 10 mm, or at least 20 mm. In yet other embodiments, the width of fluid channels may at most 20 mm, at most 10 mm, at most 5 mm, at most 2.5 mm, at most 1 mm, at most 750 micrometers, at most 500 micrometers, at most 400 micrometers, at most 300 micrometers, at most 200 micrometers, at most 100 micrometers, or at most 50 micrometers. The width of fluid channels can be about 2 mm. The width of the fluid channels may fall within any range bounded by any of these values (e.g. from about 250 μm to about 3 mm).

The fluid channels in the cartridge can have a depth. The depth of the fluid channels in cartridge designs can be between 50 micrometers and 2 mm. The depth of fluid channels may be at least 50 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, at least 400 micrometers, at least 500 micrometers, at least 750 micrometers, at least 1 mm, at least 1.25 mm, at least 1.5 mm, at least 1.75 mm, or at least 2 mm. The depth of fluid channels may at most 2 mm, at most 1.75 mm, at most 1.5 mm, at most 1.25 mm, at most 1 mm, at most 750 micrometers, at most 500 micrometers, at most 400 micrometers, at most 300 micrometers, at most 200 micrometers, at most 100 micrometers, or at most 50 micrometers. The depth of the fluid channels can be about 1 mm. The depth of the fluid channels may fall within any range bounded by any of these values (e.g. from about 800 micrometers to about 1 mm).

Cartridges can be fabricated using a variety of techniques and materials known to those of skill in the art. In general, the cartridges will be fabricated as a series of separate component parts and subsequently assembled using any of a number of mechanical assembly or bonding techniques. Examples of suitable fabrication techniques include, but are not limited to, conventional machining, CNC machining, injection molding, thermoforming, and 3D printing. Once the cartridge components have been fabricated they may be mechanically assembled using screws, clips, and the like, or permanently bonded using any of a variety of techniques (depending on the choice of materials used), for example, through the use of thermal bonding/welding or any of a variety of adhesives or adhesive films, including epoxy-based, acrylic-based, silicone-based, UV curable, polyurethane-based, or cyanoacrylate-based adhesives.

Cartridge components can be fabricated using any of a number of suitable materials, including but not limited to silicon, fused-silica, glass, any of a variety of polymers, e.g. polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxy resins, non-stick materials such as teflon (PTFE), metals (e.g. aluminum, stainless steel, copper, nickel, chromium, and titanium), or any combination thereof.

The inlet and outlet features of the cartridge can be designed to provide convenient and leak-proof fluid connections with the instrument, or may serve as open reservoirs for manual pipetting of samples and reagents into or out of the cartridge. Examples of convenient mechanical designs for the inlet and outlet port connectors can include, but are not limited to, threaded connectors, Luer lock connectors, Luer slip or “slip tip” connectors, press fit connectors, and the like. The inlet and outlet ports of the cartridge may further comprise caps, spring-loaded covers or closures, or polymer membranes that may be opened or punctured when the cartridge is positioned in the instrument, and which serve to prevent contamination of internal cartridge surfaces during storage or which prevent fluids from spilling when the cartridge is removed from the instrument. The one or more outlet ports of the cartridge may further comprise a removable sample collection chamber that is suitable for interfacing with stand-alone PCR thermal cyclers or sequencing instruments.

The cartridge can include integrated miniature pumps or other fluid actuation mechanisms for control of fluid flow through the device. Examples of suitable miniature pumps or fluid actuation mechanisms can include, but are not limited to, electromechanically- or pneumatically-actuated miniature syringe or plunger mechanisms, membrane diaphragm pumps actuated pneumatically or by an external piston, pneumatically-actuated reagent pouches or bladders, or electro-osmotic pumps.

The cartridge can include miniature valves for compartmentalizing pre-loaded reagents or controlling fluid flow through the device. Examples of suitable miniature valves can include, but are not limited to, one-shot “valves” fabricated using wax or polymer plugs that can be melted or dissolved, or polymer membranes that can be punctured; pinch valves constructed using a deformable membrane and pneumatic, magnetic, electromagnetic, or electromechanical (solenoid) actuation, one-way valves constructed using deformable membrane flaps, and miniature gate valves.

The cartridge can include vents for providing an escape path for trapped air. Vents may be constructed according to a variety of techniques, for example, using a porous plug of polydimethylsiloxane (PDMS) or other hydrophobic material that allows for capillary wicking of air but blocks penetration by water.

The mechanical interface features of the cartridge can provide for easily removable but highly precise and repeatable positioning of the cartridge relative to the instrument system. Suitable mechanical interface features can include, but are not limited to, alignment pins, alignment guides, mechanical stops, and the like. The mechanical design features can include relief features for bringing external apparatus, e.g. magnets or optical components, into close proximity with the microwell array chamber.

The cartridge can also include temperature control components or thermal interface features for mating to external temperature control modules. Examples of suitable temperature control elements can include, but are not limited to, resistive heating elements, miniature infrared-emitting light sources, Peltier heating or cooling devices, heat sinks, thermistors, thermocouples, and the like. Thermal interface features can be fabricated from materials that are good thermal conductors (e.g. copper, gold, silver, etc.) and can comprise one or more flat surfaces capable of making good thermal contact with external heating blocks or cooling blocks.

The cartridge can include optical interface features for use in optical imaging or spectroscopic interrogation of the microwell array. The cartridge can include an optically transparent window, e.g. the microwell substrate itself or the side of the flow cell or microarray chamber that is opposite the microwell array, fabricated from a material that meets the spectral requirements for the imaging or spectroscopic technique used to probe the microwell array. Examples of suitable optical window materials can include, but are not limited to, glass, fused-silica, polymethylmethacrylate (PMMA), polycarbonate (PC), cyclic olefin polymers (COP), or cyclic olefin copolymers (COC).

In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. 

What is claimed is:
 1. A method of barcoding a plurality of partitions comprising: introducing m component barcodes in each of the plurality of partitions, wherein the m component barcodes are selected from a set of n×m different component barcodes, wherein n and m are positive integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof; and connecting the m component barcodes to generate a combinatorial barcode, whereby each of the plurality of partitions is associated with a unique combinatorial barcode comprising the m component barcodes.
 2. The method of claim 1, wherein each of the plurality of the partitions comprises a sample.
 3. The method of claim 2, wherein the sample is a DNA sample or a RNA sample.
 4. The method of claim 2, wherein the sample is a single cell.
 5. The method of claim 1, wherein the total number of different linker sequences is m−1 or m−2.
 6. The method of claim 2, comprising hybridizing one or two of the m component barcodes to a target of the sample.
 7. The method of claim 6, comprising extending the one or two of the m component barcodes hybridized to the DNA target to produce an extension product comprising the combinatorial barcode.
 8. The method of claim 7, comprising amplifying the extension product.
 9. The method of claim 8, comprising pooling the amplification products from the plurality of partitions.
 10. The method of claim 9, comprising sequencing the pooled amplification products.
 11. The method of claim 10, comprising assembling the sequences using the combinatorial barcodes.
 12. The method of claim 11, comprising generating haplotypes using the assembled sequences.
 13. The method of claim 1, wherein the component barcodes are introduced in each of the plurality of partitions using an inkjet printer.
 14. A method of single cell sequencing comprising: depositing a first oligonucleotide comprising a first barcode selected from a first set of 40 unique barcodes and a first primer into a plurality of partitions comprising a single cell; depositing a second oligonucleotide comprising a second barcode selected from a second set of 40 unique barcodes and a second primer into the plurality of partitions comprising the single cell; contacting the first oligonucleotide with a target from the single cell; extending the first oligonucleotide to generate a first strand comprising the first barcode; contacting the second oligonucleotide with the first strand; and extending the second oligonucleotide to generate a second strand comprising the first barcode and the second barcode, whereby the target from the single cell is labeled with the first barcode and the second barcode.
 15. The method of claim 14, comprising amplifying the second strand to produce amplification products.
 16. The method of claim 15, comprising pooling the amplification products from the plurality of partitions.
 17. The method of claim 16, comprising sequencing the pooled amplification products.
 18. The method of claim 16, comprising assembling the sequences from a single cell using the first barcode and the second barcode.
 19. The method of claim 14, wherein the target is an mRNA or a DNA.
 20. The method of claim 14, wherein the first primer is an oligo-dT primer. 